Methods of preparing nucleic acids for sequencing

ABSTRACT

Aspects of the technology disclosed herein relate to methods for preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.

RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of co-pendingU.S. patent application Ser. No. 14/605,363 filed Jan. 26, 2015, whichclaims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication 61/931,959 filed Jan. 27, 2014, the entire contents of whichare incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 13, 2015, isnamed 030258-079365-US_SL.txt and is 31,392 bytes in size.

TECHNICAL FIELD

The technology described herein relates to methods of preparing andanalyzing nucleic acids.

BACKGROUND

Target enrichment prior to next-generation sequencing is morecost-effective than whole genome, whole exome, and whole transcriptomesequencing and therefore more practical for broad implementation; bothfor research discovery and clinical applications. For example, highcoverage depth afforded by target enrichment approaches enables a widerdynamic range for allele counting (in gene expression and copy numberassessment) and detection of low frequency mutations, a critical featurefor evaluating somatic mutations in cancer. Examples of currentenrichment protocols for next generation sequencing includehybridization-based capture assays (TruSeq Capture, Illumina; SureSelectHybrid Capture, Agilent) and polymerase chain reaction (PCR)-basedassays (HaloPlex, Agilent; AmpliSeq, Ion Torrent; TruSeq Amplicon,Illumina; emulsion/digital PCR, Raindance). Hybridization-basedapproaches capture not only the targeted sequences covered by thecapture probes but also near off-target bases that consume sequencingcapacity. In addition, these methods are relatively time-consuming,labor-intensive, and suffer from a relatively low level of specificity.

SUMMARY

Aspects of the technology disclosed herein relate to methods forpreparing and analyzing nucleic acids. In some embodiments, methods forpreparing nucleic acids for sequence analysis (e.g., usingnext-generating sequencing) are provided herein. In some embodiments,technology described herein is directed to methods of determiningnucleotide sequences of nucleic acids. In some embodiments, the methodsdescribed herein relate to enriching target nucleic acids prior tosequencing.

Aspects of the technology disclosed herein relate to methods ofdetermining the nucleotide sequence contiguous to a known targetnucleotide sequence. In some embodiments, the methods involve (a)contacting a target nucleic acid molecule comprising the known targetnucleotide sequence with an initial target-specific primer underhybridization conditions; (b) performing a template-dependent extensionreaction that is primed by a hybridized initial target-specific primerand that uses the target nucleic acid molecule as a template; (c)contacting the product of step (b) with a population of tailed randomprimers under hybridization conditions; (d) performing atemplate-dependent extension reaction that is primed by a hybridizedtailed random primer and that uses the portion of the target nucleicacid molecule downstream of the site of hybridization as a template; (e)amplifying a portion of the target nucleic acid molecule and the tailedrandom primer sequence with a first tail primer and a firsttarget-specific primer; (f) amplifying a portion of the ampliconresulting from step (e) with a second tail primer and a secondtarget-specific primer; and (g) sequencing the amplified portion fromstep (f) using a first and second sequencing primer. In someembodiments, the population of tailed random primers comprisessingle-stranded oligonucleotide molecules having a 5′ nucleic acidsequence identical to a first sequencing primer and a 3′ nucleic acidsequence comprising from about 6 to about 12 random nucleotides. In someembodiments, the first target-specific primer comprises a nucleic acidsequence that can specifically anneal to the known target nucleotidesequence of the target nucleic acid at the annealing temperature. Insome embodiments, the second target-specific primer comprises a 3′portion comprising a nucleic acid sequence that can specifically annealto a portion of the known target nucleotide sequence comprised by theamplicon resulting from step (e), and a 5′ portion comprising a nucleicacid sequence that is identical to a second sequencing primer and thesecond target-specific primer is nested with respect to the firsttarget-specific primer. In some embodiments, the first tail primercomprises a nucleic acid sequence identical to the tailed random primer.In some embodiments, the second tail primer comprises a nucleic acidsequence identical to a portion of the first sequencing primer and isnested with respect to the first tail primer.

In some embodiments, the methods involve (a) contacting a target nucleicacid molecule comprising the known target nucleotide sequence with apopulation of tailed random primers under hybridization conditions; (b)performing a template-dependent extension reaction that is primed by ahybridized tailed random primer and that uses the portion of the targetnucleic acid molecule downstream of the site of hybridization as atemplate; (c) contacting the product of step (b) with an initialtarget-specific primer under hybridization conditions; (d) performing atemplate-dependent extension reaction that is primed by a hybridizedinitial target-specific primer and that uses the target nucleic acidmolecule as a template; (e) amplifying a portion of the target nucleicacid molecule and the tailed random primer sequence with a first tailprimer and a first target-specific primer; (f) amplifying a portion ofthe amplicon resulting from step (e) with a second tail primer and asecond target-specific primer; and (g) sequencing the amplified portionfrom step (f) using a first and second sequencing primer. In someembodiments, the population of tailed random primers comprisessingle-stranded oligonucleotide molecules having a 5′ nucleic acidsequence identical to a first sequencing primer and a 3′ nucleic acidsequence comprising from about 6 to about 12 random nucleotides. In someembodiments, the first target-specific primer comprises a nucleic acidsequence that can specifically anneal to the known target nucleotidesequence of the target nucleic acid at the annealing temperature. Insome embodiments, the second target-specific primer comprises a 3′portion comprising a nucleic acid sequence that can specifically annealto a portion of the known target nucleotide sequence comprised by theamplicon resulting from step (c), and a 5′ portion comprising a nucleicacid sequence that is identical to a second sequencing primer and thesecond target-specific primer is nested with respect to the firsttarget-specific primer. In some embodiments, the first tail primercomprises a nucleic acid sequence identical to the tailed random primer.In some embodiments, the second tail primer comprises a nucleic acidsequence identical to a portion of the first sequencing primer and isnested with respect to the first tail primer.

In some embodiments, the methods further involve a step of contactingthe sample and/or products with RNase after extension of the initialtarget-specific primer. In some embodiments, the tailed random primercan form a hair-pin loop structure. In some embodiments, the initialtarget-specific primer and the first target-specific primer areidentical. In some embodiments, the tailed random primer furthercomprises a barcode portion comprising 6-12 random nucleotides betweenthe 5′ nucleic acid sequence identical to a first sequencing primer andthe 3′ nucleic acid sequence comprising 6-12 random nucleotides.

In some embodiments, the methods involve (a) contacting a target nucleicacid molecule comprising the known target nucleotide sequence with apopulation of tailed random primers under hybridization conditions; (b)performing a template-dependent extension reaction that is primed by ahybridized tailed random primer and that uses the portion of the targetnucleic acid molecule downstream of the site of hybridization as atemplate; (c) amplifying a portion of the target nucleic acid moleculeand the tailed random primer sequence with a first tail primer and afirst target-specific primer; (d) amplifying a portion of the ampliconresulting from step (c) with a second tail primer and a secondtarget-specific primer; and (e) sequencing the amplified portion fromstep (d) using a first and second sequencing primer. In someembodiments, the population of tailed random primers comprisessingle-stranded oligonucleotide molecules having a 5′ nucleic acidsequence identical to a first sequencing primer; a middle barcodeportion comprising; and a 3′ nucleic acid sequence comprising from about6 to about 12 random nucleotides. In some embodiments, the firsttarget-specific primer comprises a nucleic acid sequence that canspecifically anneal to the known target nucleotide sequence of thetarget nucleic acid at the annealing temperature. In some embodiments,the second target-specific primer comprises a 3′ portion comprising anucleic acid sequence that can specifically anneal to a portion of theknown target nucleotide sequence comprised by the amplicon resultingfrom step (c), and a 5′ portion comprising a nucleic acid sequence thatis identical to a second sequencing primer and the secondtarget-specific primer is nested with respect to the firsttarget-specific primer. In some embodiments, the first tail primercomprises a nucleic acid sequence identical to the tailed random primer.In some embodiments, the second tail primer comprises a nucleic acidsequence identical to a portion of the first sequencing primer and isnested with respect to the first tail primer. In some embodiments, theeach tailed random primer further comprises a spacer nucleic acidsequence between the 5′ nucleic acid sequence identical to a firstsequencing primer and the 3′ nucleic acid sequence comprising about 6 toabout 12 random nucleotides. In certain embodiments, the unhybridizedprimers are removed from the reaction after an extension step. In someembodiments, the second tail primer is nested with respect to the firsttail primer by at least 3 nucleotides. In certain embodiments, the firsttarget-specific primer further comprises a 5′ tag sequence portioncomprising a nucleic acid sequence of high GC content which is notsubstantially complementary to or substantially identical to any otherportion of any of the primers. In some embodiments, the second tailprimer is identical to the full-length first sequencing primer. Incertain embodiments, the portions of the target-specific primers thatspecifically anneal to the known target will anneal specifically at atemperature of about 65° C. in a PCR buffer. In some embodiments, thesample comprises genomic DNA. In some embodiments, the sample comprisesRNA and the method further comprises a first step of subjecting thesample to a reverse transcriptase regimen. In certain embodiments, thenucleic acids present in the sample have not been subjected to shearingor digestion. In some embodiments, the sample comprises single-strandedgDNA or cDNA. In certain embodiments, the reverse transcriptase regimencomprises the use of random hexamers. In some embodiments, a generearrangement comprises the known target sequence. In certainembodiments, the gene rearrangement is present in a nucleic acidselected from the group consisting of: genomic DNA; RNA; and cDNA. Insome embodiments, the gene rearrangement comprises an oncogene. Incertain embodiments, the gene rearrangement comprises a fusion oncogene.In some embodiments, the nucleic acid product is sequenced by anext-generation sequencing method. In certain embodiments, thenext-generation sequencing method comprises a method selected from thegroup consisting of: Ion Torrent, Illumina, SOLiD, 454; MassivelyParallel Signature Sequencing solid-phase, reversible dye-terminatorsequencing; and DNA nanoball sequencing. In certain embodiments, thefirst and second sequencing primers are compatible with the selectednext-generation sequencing method. In some embodiments, the methodcomprises contacting the sample, or separate portions of the sample,with a plurality of sets of first and second target-specific primers. Incertain embodiments, the method comprises contacting a single reactionmixture comprising the sample with a plurality of sets of first andsecond target-specific primers. In some embodiments, the plurality ofsets of first and second target-specific primers specifically anneal toknown target nucleotide sequences comprised by separate genes. Incertain embodiments, at least two sets of first and secondtarget-specific primers specifically anneal to different portions of aknown target nucleotide sequence. In some embodiments, at least two setsof first and second target-specific primers specifically anneal todifferent portions of a single gene comprising a known target nucleotidesequence. In certain embodiments, at least two sets of first and secondtarget-specific primers specifically anneal to different exons of a genecomprising a known nucleotide target sequence. In some embodiments, theplurality of first target-specific primers comprise identical 5′ tagsequence portions. In certain embodiments, each tailed random primer ina population of tailed random primers further comprises an identicalsample barcoding portion. In some embodiments, multiple samples are eachcontacted with a separate population of tailed random primers with asample barcoding portion; wherein each population of tailed randomprimers has a distinct sample barcoding portion; and wherein the samplesare pooled after step (b). In certain embodiments, each amplificationstep comprises a set of cycles of a PCR amplification regimen from 5cycles to 20 cycles in length. In some embodiments, the target-specificprimers and the tail primers are designed such that they willspecifically anneal to their complementary sequences at an annealingtemperature of from about 61 to 72° C. In some embodiments, thetarget-specific primers and the tail primers are designed such that theywill specifically anneal to their complementary sequences at anannealing temperature of about 65° C. In certain embodiments, the targetnucleic acid molecule is from a sample, optionally which is a biologicalsample obtained from a subject. In some embodiments, the sample isobtained from a subject in need of treatment for a disease associatedwith a genetic alteration. In certain embodiments, the disease iscancer. In some embodiments, the sample comprises a population of tumorcells. In certain embodiments, the sample is a tumor biopsy. In someembodiments, the cancer is lung cancer. In certain embodiments, adisease-associated gene comprises the known target sequence. In someembodiments, a gene rearrangement product in the sample comprises theknown target sequence. In certain embodiments, the gene rearrangementproduct is an oncogene.

Aspects of the technology disclosed herein relate to methods ofpreparing nucleic acids for analysis. In some embodiments, the methodsinvolve method (a) contacting a nucleic acid template comprising a firststrand of a target nucleic acid with a complementary target-specificprimer that comprises a target-specific hybridization sequence, underconditions to promote template-specific hybridization and extension ofthe target-specific primer; and (b) contacting a nucleic acid templatecomprising a second strand that is complementary to the first strand ofthe target nucleic acid with a plurality of different primers that sharea common sequence that is 5′ to different hybridization sequences, underconditions to promote template-specific hybridization and extension ofat least one of the plurality of different primers, in which anextension product is generated to contain both a sequence that ischaracteristic of the target-specific primer and a sequence that ischaracteristic of the at least one of the plurality of differentprimers. In some embodiments, the target nucleic acid is a ribonucleicacid. In certain embodiments, the target nucleic acid is adeoxyribonucleic acid. In some embodiments, steps (a) and (b) areperformed sequentially. In certain embodiments, the nucleic acidtemplate in step (a) comprises an extension product resulting from thehybridization and extension of the at least one of the plurality ofdifferent primers in step (b). In some embodiments, the nucleic acidtemplate in step (b) comprises an extension product resulting from thehybridization and extension of the target-specific primer in step (a).In certain embodiments, the target nucleic acid is a messenger RNAencoded from a chromosomal segment that comprises a geneticrearrangement. In some embodiments, the target nucleic acid is achromosomal segment that comprises a portion of a genetic rearrangement.In certain embodiments, the genetic rearrangement is an inversion,deletion, or translocation. In some embodiments, the methods furtherinvolve amplifying the extension product. In certain embodiments, themethods further involve contacting the extension product or amplifiedextension product with an immobilized oligonucleotide under conditionsin which hybridization occurs between the extension product andimmobilized oligonucleotide. In certain embodiments, the target nucleicacid comprises a target portion having a known sequence and a flankingportion having an unknown sequence. In some embodiments, differenthybridization sequences are complementary to the flanking portion. Incertain embodiments, the target-specific hybridization sequence iscomplementary to the target portion. In some embodiments, thetarget-specific primer further comprises, 5′ to the target-specifichybridization sequence, at least one of an index sequence, a barcodesequence and an adaptor sequence. In certain embodiments, the commonsequence comprises at least one of an index sequence, barcode sequenceand an adaptor sequence. In some embodiments, the adaptor sequence is acleavable adaptor sequence for immobilizing oligonucleotides in a flowcell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict non-limiting embodiments of a work flow foramplifying and sequencing target nucleic acids that are flanked by a 3′unknown fusion partner, as described herein.

FIGS. 2A and 2B depict non-limiting embodiments of a work flow foramplifying sequencing target nucleic acids that are flanked by a 5′unknown fusion partner, as described herein.

FIG. 3 depicts a non-limiting embodiment of a work flow for amplifyingsequencing target nucleic acids that are flanked by a 3′ unknown fusionpartem using an oligonucleotide comprising a hairpin, as describedherein.

DETAILED DESCRIPTION

Aspects of the technology disclosed herein relate to methods forpreparing and analyzing nucleic acids. In some embodiments, methodsprovided herein are useful for determining unknown nucleotide sequencescontiguous to (adjacent to) a known target nucleotide sequence.Traditional sequencing methods generate sequence information randomly(e.g. “shotgun” sequencing) or between two known sequences which areused to design primers. In contrast, methods described herein, in someembodiments, allow for determining the nucleotide sequence (e.g.sequencing) upstream or downstream of a single region of known sequencewith a high level of specificity and sensitivity. Accordingly, in someembodiments, methods provided herein are useful for determining thesequence of fusions (e.g., fusion mRNAs) that result from genearrangements (e.g., rearrangements that give rise to cancer or otherdisorders). In some embodiments, methods provided herein for preparingnucleic acids for analysis (e.g., for sequencing) involve a first roundof extension using a target-specific primer that targets a knownsequence of a target nucleic acid (e.g., a known sequence of a 1^(st)gene) followed by a second round of extension that involves the use of aheterogenous population of tailed primers (e.g., tailed random primers)that include tailed primers that have hybridization sequences that arecomplementary with an unknown sequence adjacent to the known sequence inthe target nucleic acid. In some embodiments, the tail region of tailedprimers includes barcode or index sequences that facilitate multiplexamplification and enrichment of target nucleic acids.

In some aspects of the technology disclosed herein methods are providedof preparing nucleic acids for analysis that involve (a) contacting anucleic acid template comprising a first strand of a target nucleic acidwith a complementary target-specific primer that comprises atarget-specific hybridization sequence, under conditions to promotetemplate-specific hybridization and extension of the target-specificprimer and (b) contacting a nucleic acid template comprising a secondstrand that is complementary to the first strand of the target nucleicacid with a plurality of different primers that share a common sequencethat is 5′ to different hybridization sequences, under conditions topromote template-specific hybridization and extension of at least one ofthe plurality of different primers, in which an extension product isgenerated to contain both a sequence that is characteristic of thetarget-specific primer and a sequence that is characteristic of the atleast one of the plurality of different primers. In some embodiments,steps (a) and (b) above are performed sequentially. In some embodiments,the nucleic acid template in step (a) comprises an extension productresulting from the hybridization and extension of the at least one ofthe plurality of different primers in step (b). In some embodiments, thenucleic acid template in step (b) comprises an extension productresulting from the hybridization and extension of the target-specificprimer in step (a).

In some embodiments, methods are provided for preparing nucleic acidsthat have a target region 5′ to an adjacent region (e.g., an adjacentregion of unknown sequence). In some embodiments, methods providedherein can be accomplished using one or more rounds of PCR.

For example, FIGS. 1A-B present schematics of exemplary methods ofamplifying target nucleic acids that have a known target region 5′ to anadjacent region (e.g., for purposes of sequencing the adjacent region).At step 101, initial RNA is obtained or provided in a sample and is usedas a template. RNA template is exposed to a plurality of tailed primers(e.g., tailed random primers) that comprise a common sequence that is 5′to different hybridization sequences and shared between all of thetailed primers of the population. In some embodiments, at least oneprimer hybridizes to an RNA molecule and primes a reverse transcriptasereaction to produce a complementary DNA strand. In step 102,unhybridized oligonucleotides are degraded (e.g., enzymatically, e.g.,by an exonuclease). In step 102, RNA template is degraded from thecomplementary DNA strand.

In some embodiments, a tailed primer is provided that hybridizes to thepoly-A tail of an RNA molecule. In some embodiments, the sequence of aprimer is provided that hybridizes to the poly-A tail comprises apoly-dT (e.g., a 3′ positioned stretch of 2 dTs, 3 dTs, 4 dTs, 5 dTs, 6dTs, 7 dTs, 8 dTs, 9 dTs, 10 dTs, or more). In some embodiments, aplurality of tailed primers are provided each of which comprises acommon sequence. In some embodiments, a plurality of tailed primers isprovided each of which further comprises a barcode or index sequence.

It should be appreciated that in methods disclosed herein an RNAtemplate may be degraded by any appropriate method, including, forexample, by enzymatic degradation (e.g., using RNaseH, Uracylglycosylase, etc.), by hydrolysis (e.g., by exposing the RNA torelatively high pH conditions (e.g., pH 10, pH 11, pH 12), etc. In someembodiments, hydrolyzing RNA by exposure to relatively high pH isadvantageous because it is relatively inexpensive (compared with certainother methods, e.g., certain enzymatic methods) and because it destroysboth RNA and DNA:RNA hybrids. In some embodiments, RNA is degraded byhydrolysis caused by exposure to relatively high pH conditions at arelatively high temperature (e.g., temperature greater than 60° C.(e.g., 60° C. to 95° C.)). In some embodiments, the use of relativelyhigh temperatures is advantageous because it heat inactivates enzymesused in prior preparative steps (e.g., RT enzymes). In some embodiments,an initial nucleic acid may be DNA that is obtained or provided in asample and is used as a template. In such embodiments, steps 101 and 102may be omitted.

In step 103, DNA molecules produced by reverse transcription arecontacted by one or more initial target-specific primers which may ormay not be the same as the first target-specific primer. In step 104,hybridization of the initial target-specific primer to a portion of thetarget nucleic acid (the “target sequence”) primes an extension reactionusing a DNA molecule as a template to produce a complementary DNAstrand. Extension products are purified in step 105. However, in someembodiments, DNA produced in step 104 may be amplified directly, e.g.,by PCR, without purification.

In step 106, DNA molecules are contacted by a first target-specificprimer and a first tail primer. The first target-specific primerhybridizes to a portion of the target nucleic acid. In some embodiments,pools of different first target-specific primers can be used thathybridize to different portions of a target nucleic acid. In someembodiments, use of different target specific primers can be advantegousbecause it allows for generation of different extension products havingoverlapping but staggered sequences relative to a target nucleic acid.In some embodiments, different extension products can be sequenced toproduce overlapping sequence reads. In some embodiments, overlappingsequence reads can be evaluated to assess accuracy of sequenceinformation, fidelity of nucleic acid amplification, and/or to increaseconfidence in detecting mutations, such as detecting locations ofchromosomal rearrangements (e.g., fusion breakpoints). In someembodiments, pools of different first target-specific primers can beused that hybridize to different portions of different target nucleicacids present in sample. In some embodiments, use of pools of differenttarget-specific primers is advantageous because it facilitatesprocessing (e.g., amplification) and analysis of different targetnucleic acids in parallel. In some embodiments, up to 2, up to 3, up to4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, upto 20, up to 100 or more pools of different first target-specificprimers are used. In some embodiments, 2 to 5, 2 to 10, 5 to 10, 5 to15, 10 to 15, 10 to 20, 10 to 100, 50 to 100, or more pools of differentfirst target-specific primers are used.

In FIGS. 1A and 1B, a first tail primer hybridizes to at least a portionof a DNA molecule provided by the tail portion of the tailed primer ofstep 101. In some embodiments, the first tail primer hybridizes to thecommon sequence provided by the tail of the one or more primers of step101. In some embodiments, a nested target specific primer (nested withrespect to the target specific primer of step 103) is used in step 106.In some embodiments, a first tail primer may comprise an additionalsequence 5′ to the hybridization sequence that may include barcode,index, adapter sequences, or sequencing primer sites, for example. Instep 107, hybridization of the first target-specific primer and thefirst tail nucleic acid molecule allows for amplification of a productin a polymerase chain reaction (PCR). In some embodiments, amplifiedproducts are purified in step 108.

In some embodiments, the ssDNA product of step 103 is amplified directly(e.g., by PCR) rather than performing the extension reaction of step 104and purification of step 105. Similarly, in some embodiments of any ofthe methods disclosed herein, ssDNA products may be amplified directly(e.g., by PCR) rather than performing an extension reaction to producedsDNA prior to purification and/or PCR. In some embodiments, first andsecond tail primers can be incorporated during a PCR. In someembodiments, primers (e.g., first tail primers, target specific primers)are used in a PCR or extension reaction and then excess primer isremoved using a single stranded nuclease. Subsequent rounds of PCR orextension may be performed using different primers (e.g., second tailprimers or nested primers or a second target specific primer) toincorporate different sequences into the resulting products.

In some embodiments, an exonuclease (e.g., ExoI) may be used to degradesingle-stranded DNA. In some embodiments, an exonuclease is used todegrade ssDNA and the amplified product is processed directly accordingto steps 109 _(A-B) and 110 _(A-B), without purification at step 108.

In FIG. 1A, at step 109 _(A), amplified DNA products of step 107 (e.g.,as purified in step 108) are contacted with a second target-specificprimer and a second tail primer. In some embodiments, the secondtarget-specific primer hybridizes to a sequence that is present withinthe template DNA molecule 3′ of the sequence of the firsttarget-specific primer such that the reactions are nested. In someembodiments, nesting of the second target-specific primer relative tothe first target-specific primer may improve specificity of thehybridization reaction. In some embodiments, the second target-specificprimer may comprise an additional sequence 5′ to the hybridizationsequence that may include barcode, index, adapter sequences, orsequencing primer sites, for example. In step 110 _(A), the amplifiedDNA products of step 107 (e.g., as purified in step 108) are amplifiedby PCR in which the extensions are primed by the second target-specificprimer and a second tail primer. In some embodiments, a portion of theamplified product from step 107 is further amplified. In someembodiments, a third primer is used that hybridizes to the common tailin the second target specific primer and adds additional sequences suchas a barcodes, adapters, etc.

In some embodiments, the second target-specific primer comprises anucleotide sequence 5′ to the target-specific sequence that comprises abarcode, index, or adapter sequences. In some embodiments, the secondtail primer hybridizes to a sequence that is present within the templateDNA molecule 3′ of the sequence of the first tail primer such that thereactions are nested. In such embodiments, a portion of the product fromstep 106 is amplified. In some embodiments, the second tail primer maycomprise additional sequences 5′ to the hybridization sequence that mayinclude barcode, index, adapter sequences or sequencing primer sites.Hybridization of the second target-specific primer and the second tailprimer allows for exponential amplification of a portion of the targetnucleic acid molecule in a PCR reaction.

In some embodiment, the first target-specific primer of step 106 may beused with a second target-specific primer. In such embodiments,hybridization of the first target-specific primer and the secondtarget-specific primer allows for amplification of product in apolymerase chain reaction (PCR). In some embodiments, steps 108-110 maybe omitted. The amplification products are purified in reaction 111 andready for analysis. For example, products purified in step 111 can besequenced (e.g., using a next generation sequencing platform.) In someembodiments the first target-specific primer or second target-specificprimer may comprise additional sequences 5′ to a hybridization sequencethat may include barcode, index, adapter sequences or sequencing primersites.

In some embodiments, as depicted in FIG. 1B, in step 109 _(B), DNAproducts of step 107 (e.g., as purified in step 108) are contacted witha second target-specific primer and a second tail primer. The secondtarget-specific primer is further contacted by an additional primer(e.g., a primer having 3′ sequencing adapter/index sequences) thathybridizes with the common sequence of the second target-specificprimer. In some embodiments the additional primer may compriseadditional sequences 5′ to the hybridization sequence that may includebarcode, index, adapter sequences or sequencing primer sites. In someembodiments, the additional primer is a generic sequencing adapter/indexprimer. In some embodiments, the second target-specific primer may benested relative to the target-specific primer used in step 107. In step110 _(B), the DNA products of step 107 (e.g., as purified in step 108)are amplified by PCR in which the extensions are primed by the secondtarget-specific primer and a second tail primer. Hybridization of thesecond target-specific primer, the additional primer, and the secondtail primer allows for exponential amplification of a portion of thetarget nucleic acid molecule in a PCR reaction. In such embodiments, aportion of the amplified product from step 108 is amplified.

In some embodiment, the first tail primer of step 106 may be used with asecond target-specific primer. In such embodiments, hybridization of thefirst tail primer and the second target-specific primer allows foramplification of product in a polymerase chain reaction (PCR),optionally with an additional primer (e.g., a primer having 3′sequencing adapter/index sequences) that hybridizes with the commonsequence of the second target-specific primer. In some embodiments,steps 108-110 may be omitted.

The products are purified in reaction 111 and ready for analysis. Forexample, products purified in step 111 can be sequenced (e.g., using anext generation sequencing platform).

In some embodiments, steps 101-104, 106-107, and 109-110 are performedconsecutively in a single reaction tube without any interveningpurification steps. In some embodiments, all of the components involvedin steps 101-104, 106-107, 109-110 are present at the outset andthroughout the reaction. In some embodiments, steps 101-104 areperformed consecutively in a single reaction tube. In some embodiments,all of the components involved in steps 101-104 are present at theoutset and throughout the reaction. In some embodiments, steps 106-107are performed consecutively in a single reaction tube. In someembodiments, all of the components involved in steps 106-107 are presentat the outset and throughout the reaction. In some embodiments, steps109 _(A)-110 _(A) or 109 _(B)-110 _(B) are performed consecutively in asingle reaction tube. In some embodiments, all of the componentsinvolved in steps 109 _(A)-110 _(A) or 109 _(B)-110 _(B) are present atthe outset and throughout the reaction.

In some embodiments, methods are provided for preparing nucleic acidsthat have a target region 3′ to an adjacent region (e.g., an adjacentregion of unknown sequence content). For example, FIG. 2 presents aschematic of an exemplary method of amplifying and sequencing targetnucleic acids that have a known target region 3′ to an adjacent region.In FIGS. 2A-B, an initial RNA (e.g., a fusion mRNA) is obtained orprovided in a sample and is used as a template for the proceedingmethod. At step 201, the RNA template is exposed to one or more initialtarget-specific primers that hybridize to one or more target nucleotidesequences and function to prime a reverse transcription reaction suchthat a complementary DNA molecule is produced using the initial RNA as atemplate. In some embodiments, an initial target-specific primerhybridizes to the poly-A tail of an RNA template. In some embodiments,the sequence of the primer that hybridizes to the poly-A tail comprisesa poly-dT (e.g., a 3′ positioned stretch of 2 dTs, 3 dTs, 4 dTs, 5 dTs,6 dTs, 7 dTs, 8 dTs, 9 dTs, 10 dTs, or more). In step 202 theunhybridized primers are degraded (e.g., enzymatically, e.g., by anexonuclease). In step 202, RNA template is degraded from thecomplementary DNA strand (e.g., enzymatically, e.g., by RNaseH).

In step 203, DNA molecules that were generated by reverse transcriptionare contacted by a heterogenous population of tailed primers (e.g.,tailed random primers). In some embodiments, the tail portion of each ofthe tailed primers is a shared or common sequence, identical between allprimers of the population of tailed primers. In some embodiments, atleast one primer comprises a hybridization sequence that iscomplementary to and hybridizes to the target acid template. In step204, tailed primers that are hybridized with the template nucleic acidare extended in a template-dependent extension reaction to producecomplementary DNA strands that incorporate the tailed primer sequenceand the template sequence. The resulting double stranded DNA product ispurified in step 205.

In step 206, DNA products purified in step 205 are contacted with afirst target-specific primer and a first tail primer. The firsttarget-specific primer hybridizes to a target sequence (region) of theDNA. The first tail primer hybridizes to a portion of the DNA moleculecharacteristic of the tail of the tailed primers of step 203. In someembodiments, the first tail primer hybridizes to the common sequenceprovided by the tail of the one or more primers of step 203. In someembodiments, a first tail primer comprises a barcode or index sequence.In step 207 hybridization of the first target-specific primer and thefirst tail primer facilitates exponential amplification of a portion ofthe target nucleic acid molecule in an amplification reaction (e.g., aPCR reaction). In some embodiments, the first tail primer may compriseadditional sequences 5′ to the hybridization sequence that may includebarcode, index, adapter sequences or sequencing primer sites. Amplifiedproducts are purified in step 208. In some embodiments, the purificationin step 208 is skipped. For example, in some embodiments, primers (e.g.,second target specific primers and 2^(nd) tail primers) are used in aPCR or extension reaction and then excess primer is removed using asingle stranded DNA nuclease. Subsequent rounds of PCR or extension maybe performed using different primers to incorporate different sequencesinto the resulting products.

In some embodiments, as depicted in FIG. 2A at step 209 _(A), DNAmolecules produced in step 207 (e.g., as purified in step 208) arecontacted with a second target-specific primer and a second tail primer.In some embodiments, the second target-specific primer is nestedrelative to the target specific primer used in step 207. In someembodiments, at least a portion of the product from step 209 _(A) isamplified.

In some embodiments, the second target-specific primer comprises anucleotide sequence 5′ to the target-specific sequence that comprises abarcode, index, or adapter sequences. In some embodiments, the secondtail primer hybridizes to a sequence that is present within the templateDNA molecule 3′ of the sequence of the first tail primer such that thereactions are nested. In some embodiments, at least a portion of theproduct from step 209 _(A) is amplified.

In some embodiments, first tail primer of step 206 may be used with asecond target-specific primer. In such embodiments, hybridization of thefirst tail primer and the second target-specific primer allows foramplification of product in a polymerase chain reaction (PCR). In suchembodiments, steps 208-210 may be omitted.

In some embodiments, as depicted in FIG. 2B at step 209 _(B), DNAmolecules produced in step 207 (e.g., as purified in step 208) arecontacted with a second target-specific primer and a second tail primerwherein the second target-specific primer hybridizes to a targetsequence that is present within the template DNA molecule 3′ of thesequence of the first target-specific primer such that the reactions arenested. In some embodiments, at least a portion of the product from step209 _(B) is amplified.

In some embodiments, the second target-specific primer is furthercontacted by an additional primer (e.g., a primer having 3′ sequencingadapter/index sequences) that hybridizes with the common sequence of thesecond target-specific primer. In some embodiments the additional primermay comprise additional sequences 5′ to the hybridization sequence thatmay include barcode, index, adapter sequences or sequencing primersites. In some embodiments, the additional primer is a genericsequencing adapter/index primer. In such embodiments, hybridization ofthe second target-specific primer, the additional primer, and a secondtail primer allows for exponential amplification of a portion of thetarget nucleic acid molecule in a PCR reaction.

In some embodiments, the second target-specific primer comprises anucleotide sequence 5′ to the target-specific sequence that comprises abarcode, index, or adapter sequences. In some embodiments, the secondtail primer hybridizes to a sequence that is present within the templateDNA molecule 3′ of the sequence of the first tail primer such that thereactions are nested. In some embodiments, at least a portion of theproduct from step 209 _(B) is amplified.

In some embodiments, the second tail primer may comprise additionalsequence 5′ to the hybridization sequence that may include barcode,index, adapter sequences or sequencing primer sites. In step 210,hybridization of the second target-specific primer and the second tailprimer facilitates exponential amplification of a portion of the targetnucleic acid molecule in an amplification reaction (e.g., a PCRreaction). The amplification product of step 210 is purified in reaction211 and ready for analysis. For example, products purified in step 211can be sequenced (e.g., using a next generation sequencing platform.)

In some embodiments, steps 201-204 are performed consecutively in asingle reaction tube. In some embodiments, all of the components involvein steps 201-204 are present at the outset and throughout the reaction.In some embodiments, steps 206-207 are performed consecutively in asingle reaction tube. In some embodiments, all of the components involvein steps 206-207 are present at the outset and throughout the reaction.In some embodiments, steps 209 _(A)-210 or 209 _(B)-210 are performedconsecutively in a single reaction tube. In some embodiments, all of thecomponents involve in steps 209 _(A)-210 or 209 _(B)-210 are present atthe outset and throughout the reaction.

In some embodiments, methods provided herein involve use of randomsequences as molecular barcodes. In some embodiments, molecular barcodesare built into primers (e.g., RT primers, target-specific primers,extension sequence primers) such that each individual molecule producedby a primer obtains a unique barcode tag. Thus, in some embodiments, themolecular barcode tag permits a determination of whether a sequencedmolecular is unique. In some embodiments, molecular barcodes may be usedto silence sequencing errors, improve confidence calling of fusions orother mutations, and improided detection limits.

In some embodiments, methods are provided for preparing nucleic acidsthat have a target region 5′ to an adjacent region (e.g., an adjacentregion of unknown sequence) using an oligonucleotide comprising ahairpin. In some embodiments, the oligonucleotide may have a structurethat is not a hairpin, for example, the oligonucleotide may be linear.For example, FIG. 3 presents a schematic of an exemplary method foramplifying target nucleic acids that have a known target region 5′ to anadjacent region (for purposes of sequencing the adjacent region). Atstep 301, an initial RNA is obtained or provided in a sample and is usedas a template for the proceeding method. The RNA template is exposed toa plurality of hairpin primers (e.g., random primers with a hairpintail) that comprise a hairpin sequence that is 5′ to differenthybridization sequences and shared between all of the primers of thepopulation. The hairpin sequence comprises two complementary commonsequences that flank a molecular barcode sequence (MBC). Thecomplementary common sequences base pair to form the stem-loop hairpinstructure and protect the MBC sequence. In some embodiments, theplurality of primers is in another structure (not a hairpin) andcomprises two complementary common sequences that flank a molecularbarcode sequence (MBC). In some embodiments, at least one primerhybridizes to the RNA molecule and primes a reverse transcriptasereaction to produce a complementary DNA strand. In some embodiments, aprimer hybridizes to the poly-A tail of the RNA molecule. In someembodiments, the sequence of the primer that hybridizes to the poly-Atail comprises a poly-dT (e.g., a 3′ positioned stretch of 2 dTs, 3 dTs,4 dTs, 5 dTs, 6 dTs, 7 dTs, 8 dTs, 9 dTs, 10 dTs, or more). In step 302,any unhybridized oligonucleotides are enzymatically degraded (e.g., byan exonuclease). Also in step 302, the RNA template is enzymaticallydegraded from the complementary DNA strand (e.g., by RNaseH).

In step 303, DNA molecules produced by reverse transcription arecontacted by one or more initial target-specific primers which may ormay not be the same as the first target-specific primer. In step 304,hybridization of the first target-specific primer to a portion of thetarget nucleic acid primes an extension reaction using the DNA moleculeas a template to produce a complementary DNA strand. In someembodiments, synthesis of the complementary DNA strand may reduce oreliminate hairpin formation of the complementary common sequences.Extension products are purified in step 305.

In step 306, DNA molecules are contacted by a first target-specificprimer and a tailed primer. The first target-specific primer hybridizesto a portion of the target nucleic acid. The first tail primerhybridizes to a portion of the DNA molecule provided by the commonsequence involve in hairpin formation step 301. In some embodiments, anested target-specific primer (e.g., nested with respect to thetarget-specific primer of step 303) is used in step 306. In someembodiments, the first tailed primer may comprise an additional sequence5′ to the hybridization sequence that may include barcode, index,adapter sequences, or sequencing primer sites, for example. In step 307,hybridization of each of the first target-specific primer and the tailedprimer allows for amplification of a portion of the target nucleic acidmolecule in a polymerase chain reaction (PCR). In some embodiments,amplified products are purified in step 308.

In step 309, amplified DNA products (e.g., those purified in step 308)are contacted with a second target-specific primer and a common sequenceprimer. In some embodiments, the second target-specific primerhybridizes to a sequence that is present within the template DNAmolecule 3′ of the sequence of the first target-specific primer suchthat the reactions are nested. In some embodiments, the common sequenceprimer hybridizes with a sequence provided by the first tail primer instep 306. In some embodiments, in step 310, DNA products purified instep 308 are amplified by PCR in which the extensions are primed by asecond target-specific primer and a common sequence primer. In someembodiments, amplified product from step 308 may be amplified.

In some embodiments, the second target-specific primer comprises anucleotide sequence 5′ to the target-specific sequence that comprises abarcode, index, or adapter sequences. In some embodiments, the secondtail primer hybridizes to a sequence that is present within the templateDNA molecule 3′ of the sequence of the first tail primer such that thereactions are nested. In such embodiments, a portion of the product fromstep 308 is amplified. In some embodiments, the common sequence primermay comprise additional sequences 5′ to the hybridization sequence thatmay include barcode, index, adapter sequences or sequencing primersites. Hybridization of the second target-specific primer and the commonsequence primer allows for exponential amplification of a portion of thetarget nucleic acid molecule in a PCR reaction. In some embodiments,products are purified in reaction 311 useful for analysis. For example,products purified in step 311 can be sequenced (e.g., using a nextgeneration sequencing platform.)

In some embodiments, all of the components involved in steps 301-311 arepresent at the outset and throughout the reaction. In some embodiments,steps 301-304, 306-307, and 309-310 are performed consecutively in asingle reaction tube without any intervening purification steps. In someembodiments, steps 301-304 are performed consecutively in a singlereaction tube. In some embodiments, all of the components involve insteps 301-304 are present at the outset and throughout the reaction. Insome embodiments, steps 306-307 are performed consecutively in a singlereaction tube. In some embodiments, all of the components involve insteps 306-307 are present at the outset and throughout the reaction. Insome embodiments, steps 309-310 are performed consecutively in a singlereaction tube. In some embodiments, all of the components involve insteps 309-310 are present at the outset and throughout the reaction.

In some embodiments, methods are provided herein that involvedetermining the nucleotide sequence contiguous to (adjacent to) a knowntarget nucleotide sequence. In some embodiments, methods comprisecontacting a target nucleic acid molecule comprising the known targetnucleotide sequence with an initial target-specific primer undersuitable hybridization conditions. In some embodiments, the methodsfurther comprise maintaining the target nucleic acid molecule underconditions that promote extension of the hybridized initialtarget-specific primer (e.g., using the target nucleic acid molecule asa template), thereby producing a first extension product. In someembodiments, the methods further comprise contacting the extensionproduct with a population of tailed random primers under suitablehybridization conditions. In some embodiments, the methods furthercomprise maintaining the extension product under conditions that promoteextension of a hybridized tailed random primer using the portion of thetarget nucleic acid molecule downstream of the site of hybridization asa template, thereby producing a second extension product. In someembodiments, the methods further comprise amplifying a portion of thetarget nucleic acid molecule and the tailed random primer sequence witha first tail primer and a first target-specific primer, therebyproducing a first amplicon. In some embodiments, the methods furthercomprise amplifying a portion of the amplicon with a second tail primerand a second target-specific primer, thereby producing a secondamplicon.

In some embodiments, one or more target-specific primers used in themethods may be nested with respect to one or more other target-specificprimers. For example, in some embodiments, a second target-specificprimer is internal to a first target-specific primer. In someembodiments, target-specific primers are the same. In some embodiments,target-specific primers are nested but overlapping with respect totarget complementarity. In some embodiments, target-specific primers arenested and non-overlapping. In some embodiments, combinations ofidentical and nested target specific primers are used in the same ordifferent amplification steps. In some embodiments, nesting of primersincreases target specificity. In some embodiments, the methods furthercomprise sequencing the second amplicon using a first and secondsequencing primer. In some embodiments, the population of tailed randomprimers comprises single-stranded oligonucleotide molecules having a 5′nucleotide sequence identical to a first sequencing primer and a 3′nucleotide comprising from random nucleotides (e.g., about 6 to about 12random nucleotides). In some embodiments, the first target-specificprimer comprises a nucleic acid sequence that can specifically anneal tothe known nucleotide sequence of the target nucleic acid at anappropriate annealing temperature. In some embodiments, the secondtarget-specific primer comprises a 3′ portion comprising a nucleic acidsequence that can specifically anneal to a portion of the known targetnucleotide sequence comprised by the first amplicon, and a 5′ portioncomprising a nucleic acid sequence that is identical to a secondsequencing primer and the second target-specific primer is nested withrespect to the first target-specific primer. In some embodiments, thefirst tail primer comprises a nucleic acid sequence identical to thecommon sequence of the tail of the tailed random primer. In someembodiments, the common sequence on the tailed random primer is theexact match of the common sequence on the first tail primer. In someembodiments, the second tail primer comprises a nucleic acid sequenceidentical to a portion of the first sequencing primer and is nested withrespect to the first tail primer.

As used herein, the term “target nucleic acid” refers to a nucleic acidmolecule of interest (e.g., an nucleic acid to be analzed). In someembodiments, a target nucleic acid comprises both a target nucleotidesequence (e.g., a known or predetermined nucleotide sequence) and anadjacent nucleotide sequence which is to be determined (which may bereferred to as an unknown sequence). A target nucleic acid can be of anyappropriate length. In some embodiments, a target nucleic acid isdouble-stranded. In some embodiments, the target nucleic acid is DNA. Insome embodiments, the target nucleic acid is genomic or chromosomal DNA(gDNA). In some embodiments, the target nucleic acid can becomplementary DNA (cDNA). In some embodiments, the target nucleic acidis single-stranded. In some embodiments, the target nucleic acid can beRNA, e.g., mRNA, rRNA, tRNA, long non-coding RNA, microRNA.

As used herein, the term “known target nucleotide sequence” refers to aportion of a target nucleic acid for which the sequence (e.g. theidentity and order of the nucleotide bases of the nucleic acid) isknown. For example, in some embodiments, a known target nucleotidesequence is a nucleotide sequence of a nucleic acid that is known orthat has been determined in advance of an interrogation of an adjacentunknown sequence of the nucleic acid. A known target nucleotide sequencecan be of any appropriate length.

In some embodiments, a target nucleotide sequence (e.g., a known targetnucleotide sequence) has a length of 10 or more nucleotides, 30 or morenucleotides, 40 or more nucleotides, 50 or more nucleotides, 100 or morenucleotides, 200 or more nucleotides, 300 or more nucleotides, 400 ormore nucleotides, 500 or more nucleotides. In some embodiments, a targetnucleotide sequence (e.g., a known target nucleotide sequence) has alength in range of 10 to 100 nucleotides, 10 to 500 nucleotides, 10 to1000 nucleotides, 100 to 500 nucleotides, 100 to 1000 nucleotides, 500to 1000 nucleotides, 500 to 5000 nucleotides.

In some embodiments, methods are provided herein for determiningsequences of contiguous (or adjacent) portions of a nucleic acid. Asused herein, the term “nucleotide sequence contiguous to” refers to anucleotide sequence of a nucleic acid molecule (e.g., a target nucleicacid) that is immediately upstream or downstream of another nucleotidesequence (e.g., a known nucleotide sequence). In some embodiments, anucleotide sequence contiguous to a known target nucleotide sequence maybe of any appropriate length. In some embodiments, a nucleotide sequencecontiguous to a known target nucleotide sequence comprises 1 kb or lessof nucleotide sequence, e.g. 1 kb or less of nucleotide sequence, 750 bpor less of nucleotide sequence, 500 bp or less of nucleotide sequence,400 bp or less of nucleotide sequence, 300 bp or less of nucleotidesequence, 200 bp or less of nucleotide sequence, 100 bp or less ofnucleotide sequence. In some embodiments, in which a sample comprisesdifferent target nucleic acids comprising a known target nucleotidesequence (e.g. a cell in which a known target nucleotide sequence occursmultiple times in its genome, or on separate, non-identicalchromosomes), there may be multiple sequences which comprise “anucleotide sequence contiguous to” the known target nucleotide sequence.As used herein, the term “determining a (or the) nucleotide sequence,”refers to determining the identity and relative positions of thenucleotide bases of a nucleic acid.

In some embodiments of methods disclosed herein one or more tailedrandom primers are hybridized to a nucleic acid template (e.g., atemplate comprising a strand of a target nucleic acid). In someembodiments, a target nucleic acid is present in or obtained from asample comprising a plurality of nucleic acids, one or more of whichplurality do not comprise the target nucleic acid. In some embodiments,one or more primers (e.g., one or more tailed random primers) hybridizeto substantially all of the nucleic acids in a sample. In someembodiments, one or more primers (e.g., one or more tailed randomprimers) hybridize to nucleic acids that comprise a target nucleic acidand to nucleic acids that do not comprise the target nucleotidesequence.

Aspects of certain methods disclosed herein relate to contacting anucleic acid template with a plurality of different primers that share acommon sequence that is 5′ (or upstream) to different hybridizationsequences. In some embodiments the plurality of different primers may bereferred to as a population of different primers. In some embodiments,the common sequence may be referred to as a tail, as such the primersare referred to as “tailed primers.” In some embodiments, differenthybridization sequences of a population comprise nucleotide sequencesthat occur randomly or pseudorandomly within the population. In someembodiments, nucleotide sequences that occur randomly within apopulation contain no recognizable regularities, such that, for eachnucleotide of each sequence in the population, there is an equallikelihood that the nucleotide comprises a base that is complementarywith A, T, G, or C. In such embodiments, it should be appreciated thateach nucleotide comprising a base that is complementary with A, T, G, orC may be a naturally occurring nucleotide, a non-naturally occurringnucleotide or a modified nucleotide.

As used herein, a “common sequence” or “shared sequence” refers to anucleotide sequence that is present in each nucleic acid of a populationof nucleic acids. In some embodiments, the common sequence is in a rangeof about 4 to 75, 4 to 50, 4 to 30, or 4 to 20 nucleotides in length. Insome embodiments, the common sequence is 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, or 75 nucleotides in length.

As used herein, the term “tailed random primer” refers to asingle-stranded nucleic acid molecule having a 5′ nucleotide sequence(e.g., a 5′ nucleotide sequence identical or complementary to a firstsequencing primer) and a 3′ nucleic acid sequence, in which the 3′nucleotide comprises random nucleotides (e.g., from about 3 to about 15random nucleotides, about 6 to about 12 random nucleotides). In someembodiments, the 3′ nucleotide sequence comprising random nucleotides isat least 6 nucleotides in length, e.g. 6 nucleotides or more, 7nucleotides or more, 8 nucleotides or more, 9 nucleotides or more, 10nucleotides or more, 11 nucleotides or more, 12 nucleotides or more, 13nucleotides or more, 14 nucleotides or more, 15 nucleotides or more, 20nucleotides or more, 25 nucleotides or more in length. In someembodiments, the 3′ nucleotide sequence comprising random nucleotides is3 to 6 nucleotides in length, 3 to 9 nucleotides in length, 3 to 12nucleotides in length, 5 to 9 nucleotides in length, 6 to 12 nucleotidesin length, 3 to 25 nucleotides in length, 6 to 15 nucleotides in length,or 6 to 25 nucleotides in length. In some embodiments, a tailed randomprimer can further comprise a spacer between the 5′ nucleotide sequenceand the 3′ nucleotide sequence comprising about 6 to about 12 randomnucleotides. In some embodiments, the spacer is a molecular barcode,e.g., that independently tags a template nucleic acid (e.g., a templateRNA). In some embodiments, the spacer may be 3 to 6 nucleotides inlength, 3 to 12 nucleotides in length, 3 to 25 nucleotides in length, 3to 45 nucleotides in length, 6 to 12 nucleotides in length, 8 to 16nucleotides in length, 6 to 25 nucleotides in length, or 6 to 45nucleotides in length. In some embodiments, for a populations ofprimers, the spacer is composed of random nucleotides (e.g., in whicheach of N is independently selected from A, G, C, and T). In someembodiments, the spacer (e.g., a molecular barcode (MBC)) is flanked bytwo common regions that are complementary. In some embodiments, thecomplementary common regions base pair to form the stem of a hairpinhaving a loop portion that comprises the MBC (e.g., as depicted in FIG.3). In some embodiments, this hairpin configuration protects the MBCfrom annealing to other targets inhibiting the RT reaction of theextension reaction in the case of 5′ fusions. In some embodiments, apopulation of tailed random primers can comprise individual primers withvarying 3′ sequences. In some embodiments, a population of tailed randomprimers can comprise individual primers with identical 5′ nucleotidesequences, e.g., they are all compatible with the same sequencingprimer. In some embodiments, a population of tailed random primers cancomprise individual primers with varying 5′ nucleotide sequences, e.g.an first individual primer is compatible with a first sequencing primerand a second individual primer is compatible with a second sequencingprimer.

As used herein, a “hybridization sequence” refers to a sequence of anucleic acid, such as a portion of a primer, that that has sufficientcomplementary with a sequence of another nucleic acid (e.g., a templatemolecule, a target sequence) to enable hybridization between nucleicacid. In some embodiments, the hybridization sequence is about 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or more nucleotides in length. In someembodiments, the hybridization sequence is in a range of 5 to 50nucleotides in length, 5 to 40 nucleotides in length, 5 to 35nucleotides in length, 5 to 30 nucleotides in length, 5 to 25nucleotides in length, 5 to 20 nucleotides in length, 5 to 15nucleotides in length, 5 to 10 nucleotides in length, 10 to 40nucleotides in length, 10 to 30 nucleotides in length, or 10 to 20nucleotides in length.

In some embodiments, methods described herein comprises an extensionregimen or step. In such embodiments, extension may proceed from one ormore hybridized tailed random primers, using the nucleic acid moleculeswhich the primers are hybridized to as templates. Extension steps aredescribed herein. In some embodiments, one or more tailed random primerscan hybridize to substantially all of the nucleic acids in a sample,many of which may not comprise a known target nucleotide sequence.Accordingly, in some embodiments, extension of random primers may occurdue to hybridization with templates that do not comprise a known targetnucleotide sequence.

In some embodiments, methods described herein may involve a polymerasechain reaction (PCR) amplification regimen, involving one or moreamplification cycles. As used herein, the term “amplification regimen”refers to a process of specifically amplifying (increasing the abundanceof) a nucleic acid of interest. In some embodiments, exponentialamplification occur when products of a previous polymerase extensionserve as templates for successive rounds of extension. In someembodiments, a PCR amplification regimen according to methods disclosedherein may comprise at least one, and in some cases at least 5 or moreiterative cycles. In some embodiments each iterative cycle comprisessteps of: 1) strand separation (e.g., thermal denaturation); 2)oligonucleotide primer annealing to template molecules; and 3) nucleicacid polymerase extension of the annealed primers. In should beappreciated that any suitable conditions and times involved in each ofthese steps may be used. In some embodiments, conditions and timesselected may depend on the length, sequence content, meltingtemperature, secondary structural features, or other factors relating tothe nucleic acid template and/or primers used in the reaction. In someembodiments, an amplification regimen according to methods describedherein is performed in a thermal cycler, many of which are commerciallyavailable.

In some embodiments, a nucleic acid extension reaction involves the useof a nucleic acid polymerase. As used herein, the phrase “nucleic acidpolymerase” refers an enzyme that catalyzes the template-dependentpolymerization of nucleoside triphosphates to form primer extensionproducts that are complementary to the template nucleic acid sequence. Anucleic acid polymerase enzyme initiates synthesis at the 3′ end of anannealed primer and proceeds in the direction toward the 5′ end of thetemplate. Numerous nucleic acid polymerases are known in the art andcommercially available. One group of nucleic acid polymerases arethermostable, i.e., they retain function after being subjected totemperatures sufficient to denature annealed strands of complementarynucleic acids, e.g. 94° C., or sometimes higher. A non-limiting exampleof a protocol for amplification involves using a polymerase (e.g.,Phoenix Taq, VeraSeq) under the following conditions: 98° C. for 30 s,following by 14-22 cycles comprising melting at 98° C. for 10 s,followed by annealing at 68° C. for 30 s, followed by extension at 72°C. 3 min, followed by holding of the reaction at 4° C. However, otherappropriate reaction conditions may be used. In some embodiments,annealing/extension temperatures may be adjusted to account fordifferences in salt concentration (e.g., 3° C. higher to higher saltconcentrations). In some embodiments, slowing the ramp rate (e.g., 1°C./s, 0.5° C./s, 0.28° C./s, 0.1° C./s or slower), for example, from 98°C. to 65° C., improves primer performance and coverage uniformity inhighly multiplexed samples.

In some embodiments, a nucleic acid polymerase is used under conditionsin which the enzyme performs a template-dependent extension. In someembodiments, the nucleic acid polymerase is DNA polymerase I, Taqpolymerase, Pheonix Taq polymerase, Phusion polymerase, T4 polymerase,T7 polymerase, Klenow fragment, Klenow exo-, phi29 polymerase, AMVreverse transcriptase, M-MuLV reverse transcriptase, HIV-1 reversetranscriptase, VeraSeq ULtra polymerase, VeraSeq HF 2.0 polymerase,EnzScript or another appropriate polymerase. In some embodiments, anucleic acid polymerase is not a reverse transcriptase. In someembodiments, a nucleic acid polymerase acts on a DNA template. In someembodiments, the nucleic acid polymerase acts on an RNA template. Insome embodiments, an extension reaction involves reverse transcriptionperformed on an RNA to produce a complementary DNA molecule(RNA-dependent DNA polymerase activity). In some embodiments, a reversetranscriptase is a mouse molony murine leukemia virus (M-MLV)polymerase, AMV reverse transcriptase, RSV reverse transcriptase, HIV-1reverse transcriptase, HIV-2 reverse transcriptase or anotherappropriate reverse transcriptase.

In some embodiments, a nucleic acid amplification reaction involvescycles including a strand separation step generally involving heating ofthe reaction mixture. As used herein, the term “strand separation” or“separating the strands” means treatment of a nucleic acid sample suchthat complementary double-stranded molecules are separated into twosingle strands available for annealing to an oligonucleotide primer. Insome embodiments, strand separation according to methods describedherein is achieved by heating the nucleic acid sample above its meltingtemperature (T_(m)). In some embodiments, for a sample containingnucleic acid molecules in a reaction preparation suitable for a nucleicacid polymerase, heating to 94° C. is sufficient to achieve strandseparation. In some embodiments, a suitable reaction preparationcontains one or more salts (e.g., 1 to 100 mM KCl, 0.1 to 10 MgCl₂), atleast one buffering agent (e.g., 1 to 20 mM Tris-HCL), and a carrier(e.g., 0.01 to 0.5% BSA). A non-limiting example of a suitable buffercomprises 50 mM KCl, 10 mM Tris-HCl (pH 8.8@25° C.), 0.5 to 3 mM MgCl₂,and 0.1% BSA.

In some embodiments, a nucleic acid amplification involves annealingprimers to nucleic acid templates having a strands characteristic of atarget nucleic acid. In some embodiments, a strand of a target nucleicacid can serve as a template nucleic acid.

As used herein, the term “anneal” refers to the formation of one or morecomplementary base pairs between two nucleic acids. In some embodiments,annealing involve two complementary or substantially complementarynucleic acids strands hybridizing together. In some embodiments, in thecontext of an extension reaction annealing involves the hybridize ofprimer to a template such that a primer extension substrate for atemplate-dependent polymerase enzyme is formed. In some embodiments,conditions for annealing (e.g., between a primer and nucleic acidtemplate) may vary based of the length and sequence of a primer. In someembodiments, conditions for annealing are based upon a T_(m) (e.g., acalculated T_(m)) of a primer. In some embodiments, an annealing step ofan extension regimen involves reducing the temperature following strandseparation step to a temperature based on the T_(m) (e.g., a calculatedT_(m)) for a primer, for a time sufficient to permit such annealing. Insome embodiments, a T_(m) can be determined using any of a number ofalgorithms (e.g., OLIGO™ (Molecular Biology Insights Inc. Colorado)primer design software and VENTRO NTI™ (Invitrogen, Inc. California)primer design software and programs available on the internet, includingPrimer3, Oligo Calculator, and NetPrimer (Premier Biosoft; Palo Alto,Calif.; and freely available on the world wide web (e.g., atpremierbiosoft.com/netprimer/netprlaunch/Help/xnetprlaunch.html). Insome embodiments, the T_(m) of a primer can be calculated usingfollowing formula, which is used by NetPrimer software and is describedin more detail in Frieir et al. PNAS 1986 83:9373-9377 which isincorporated by reference herein in its entirety.

T _(m) =ΔH/(ΔS+R*ln(C/4))+16.6 log([K ⁺]/(1+0.7[K ⁺]))−273.15

wherein, ΔH is enthalpy for helix formation; ΔS is entropy for helixformation; R is molar gas constant (1.987 cal/° C.*mol); C is thenucleic acid concentration; and [K⁺] is salt concentration. For mostamplification regimens, the annealing temperature is selected to beabout 5° C. below the predicted T_(m), although temperatures closer toand above the T_(m) (e.g., between 1° C. and 5° C. below the predictedT_(m) or between 1° C. and 5° C. above the predicted T_(m)) can be used,as can, for example, temperatures more than 5° C. below the predictedT_(m) (e.g., 6° C. below, 8° C. below, 10° C. below or lower). In someembodiments, the closer an annealing temperature is to the T_(m), themore specific is the annealing. In some embodiments, the time used forprimer annealing during an extension reaction (e.g., within the contextof a PCR amplification regimen) is determined based, at least in part,upon the volume of the reaction (e.g., with larger volumes involvinglonger times). In some embodiments, the time used for primer annealingduring an extension reaction (e.g., within the context of a PCRamplification regimen) is determined based, at least in part, uponprimer and template concentrations (e.g., with higher relativeconcentrations of primer to template involving less time than lowerrelative concentrations). In some embodiments, depending upon volume andrelative primer/template concentration, primer annealing steps in anextension reaction (e.g., within the context of an amplificationregimen) can be in the range of 1 second to 5 minutes, 10 seconds and 2minutes, or 30 seconds to 2 minutes. As used herein, “substantiallyanneal” refers to an extent to which complementary base pairs formbetween two nucleic acids that, when used in the context of a PCRamplification regimen, is sufficient to produce a detectable level of aspecifically amplified product.

As used herein, the term “polymerase extension” refers totemplate-dependent addition of at least one complementary nucleotide, bya nucleic acid polymerase, to the 3′ end of an primer that is anneal toa nucleic acid template. In some embodiments, polymerase extension addsmore than one nucleotide, e.g., up to and including nucleotidescorresponding to the full length of the template. In some embodiments,conditions for polymerase extension are based, at least in part, on theidentity of the polymerase used. In some embodiments, the temperatureused for polymerase extension is based upon the known activityproperties of the enzyme. In some embodiments, in which annealingtemperatures are below the optimal temperatures for the enzyme, it maybe acceptable to use a lower extension temperature. In some embodiments,enzymes may retain at least partial activity below their optimalextension temperatures. In some embodiments, a polymerase extension(e.g., performed thermostable polymerases) (e.g., Taq polymerase andvariants thereof) is performed at 65° C. to 75° C. or 68° C. to 72° C.In some embodiments, methods provided herein involve polymeraseextension of primers that are anneal to nucleic acid templates at eachcycle of a PCR amplification regimen. In some embodiments, a polymeraseextension is performed using a polymerase that has relatively strongstrand displacement activity. In some embodiments, polymerases havingstrong strand displacement are useful for preparing nucleic acids forpurposes of detecting fusions (e.g., 5′ fusions).

In some embodiments, primer extension is performed under conditions thatpermit the extension of annealed oligonucleotide primers. As usedherein, the term “conditions that permit the extension of an annealedoligonucleotide such that extension products are generated” refers tothe set of conditions including, for example temperature, salt andco-factor concentrations, pH, and enzyme concentration under which anucleic acid polymerase catalyzes primer extension. In some embodiments,such conditions are based, at least in part, on the nucleic acidpolymerase being used. In some embodiments, a polymerase may perform aprimer extension reaction in a suitable reaction preparation. In someembodiments, a suitable reaction preparation contains one or more salts(e.g., 1 to 100 mM KCl, 0.1 to 10 MgCl₂), at least one buffering agent(e.g., 1 to 20 mM Tris-HCL), a carrier (e.g., 0.01 to 0.5% BSA) and oneor more NTPs (e.g, 10 to 200 uM of each of dATP, dTTP, dCTP, and dGTP).non-limiting set of conditions is 50 mM KCl, 10 mM Tris-HCl (pH 8.8@25°C.), 0.5 to 3 mM MgCl₂, 200 uM each dNTP, and 0.1% BSA at 72° C., underwhich a polymerase (e.g., Taq polymerase) catalyzes primer extension. Insome embodiments, conditions for initiation and extension may includethe presence of one, two, three or four different deoxyribonucleosidetriphosphates (e.g., selected from dATP, dTTP, dCTP, and dGTP) and apolymerization-inducing agent such as DNA polymerase or reversetranscriptase, in a suitable buffer. In some embodiments, a “buffer” mayinclude solvents (e.g., aqueous solvents) plus appropriate cofactors andreagents which affect pH, ionic strength, etc.).

In some embodiments, nucleic acid amplification involve up to 5, up to10, up to 20, up to 30, up to 40 or more rounds (cycles) ofamplification. In some embodiments, nucleic acid amplification maycomprise a set of cycles of a PCR amplification regimen from 5 cycles to20 cycles in length. In some embodiments, an amplification step maycomprise a set of cycles of a PCR amplification regimen from 10 cyclesto 20 cycles in length. In some embodiments, each amplification step cancomprise a set of cycles of a PCR amplification regimen from 12 cyclesto 16 cycles in length. In some embodiments, an annealing temperaturecan be less than 70° C. In some embodiments, an annealing temperaturecan be less than 72° C. In some embodiments, an annealing temperaturecan be about 65° C. In some embodiments, an annealing temperature can befrom about 61 to about 72° C.

In various embodiments, methods and compositions described herein relateto performing a PCR amplification regimen with one or more of the typesof primers described herein. As used herein, “primer” refers to anoligonucleotide capable of specifically annealing to a nucleic acidtemplate and providing a 3′ end that serves as a substrate for atemplate-dependent polymerase to produce an extension product which iscomplementary to the template. In some embodiments, a primer useful inmethods described herein is single-stranded, such that the primer andits complement can anneal to form two strands. Primers according tomethods and compositions described herein may comprise a hybridizationsequence (e.g., a sequence that anneals with a nucleic acid template)that is less than or equal to 300 nucleotides in length, e.g., less thanor equal to 300, or 250, or 200, or 150, or 100, or 90, or 80, or 70, or60, or 50, or 40, or 30 or fewer, or 20 or fewer, or 15 or fewer, but atleast 6 nucleotides in length. In some embodiments, a hybridizationsequence sequence of a primer may be 6 to 50 nucleotides in length, 6 to35 nucleotides in length, 6 to 20 nucleotides in length, 10 to 25nucleotides in length.

Any suitable method may be used for synthesizing oligonucleotides andprimers. In some embodiments, commercial sources offer oligonucleotidesynthesis services suitable for providing primers for use in methods andcompositions described herein, e.g. INVITROGEN™ Custom DNA Oligos; LifeTechnologies; Grand Island, N.Y. or custom DNA Oligos from IDT;Coralville, Iowa).

In some embodiments, after an extension from a tailed random primer hasoccurred, the extension product and template can be amplified in a firstamplification step. In some embodiments, amplification may involve a setof PCR amplification cycles using a first target-specific primer and afirst tail primer. In some embodiments, the amplification may result inat least part of the tailed random primer sequence present in theextension product being amplified. In some embodiments, theamplification may result in all of the tailed random primer sequencepresent in the extension product being amplified.

As used herein, the term “first target-specific primer” refers to asingle-stranded oligonucleotide comprising a nucleic acid sequence thatcan specifically anneal under suitable annealing conditions to a nucleicacid template that has a strand characteristic of a target nucleic acid.

In some embodiments, a primer (e.g., a target specific primer) cancomprise a 5′ tag sequence portion. In some embodiments, multipleprimers (e.g., all first-target specific primers) present in a reactioncan comprise identical 5′ tag sequence portions. In some embodiments, ina multiplex PCR reaction, different primer species can interact witheach other in an off-target manner, leading to primer extension andsubsequently amplification by DNA polymerase. In such embodiments, theseprimer dimers tend to be short, and their efficient amplification canovertake the reaction and dominate resulting in poor amplification ofdesired target sequence. Accordingly, in some embodiments, the inclusionof a 5′ tag sequence in primers (e.g., on target specific primer(s)) mayresult in formation of primer dimers that contain the same complementarytails on both ends. In some embodiments, in subsequent amplificationcycles, such primer dimers would denature into single-stranded DNAprimer dimers, each comprising complementary sequences on their two endswhich are introduced by the 5′ tag. In some embodiments, instead ofprimer annealing to these single stranded DNA primer dimers, anintra-molecular hairpin (a panhandle like structure) formation may occurdue to the proximate accessibility of the complementary tags on the sameprimer dimer molecule instead of an inter-molecular interaction with newprimers on separate molecules. Accordingly, in some embodiments, theseprimer dimers may be inefficiently amplified, such that primers are notexponentially consumed by the dimers for amplification; rather thetagged primers can remain in high and sufficient concentration fordesired specific amplification of target sequences. In some embodiments,accumulation of primer dimers may be undesirable in the context ofmultiplex amplification because they compete for and consume otherreagents in the reaction.

In some embodiments, a 5′ tag sequence can be a GC-rich sequence. Insome embodiments, a 5′ tag sequence may comprise at least 50% GCcontent, at least 55% GC content, at least 60% GC content, at least 65%GC content, at least 70% GC content, at least 75% GC content, at least80% GC content, or higher GC content. In some embodiments, a tagsequence may comprise at least 60% GC content. In some embodiments, atag sequence may comprise at least 65% GC content.

In some embodiments, a target-specific primer (e.g., a secondtarget-specific primer) is a single-stranded oligonucleotide comprisinga 3′ portion comprising a nucleic acid sequence that can specificallyanneal to a portion of a known target nucleotide sequence of an ampliconof an amplification reaction, and a 5′ portion comprising a tag sequence(e.g., a nucleotide sequence that is identical to or complementary to asequencing primer (e.g., a second sequencing primer).

In some embodiments, a second target-specific primer of an amplificationregimen is nested with respect to a first target-specific primer of theamplification regimen. In some embodiments, the second target-specificprimer is nested with respect to the first target-specific primer by atleast 3 nucleotides, e.g. by 3 or more, 4 or more, 5 or more, 6 or more,7 or more, 8 or more, 9 or more, 10 or more, or 15 or more nucleotides.In some embodiments, all of the target-specific primers (e.g., secondtarget-specific primers) used in an amplification regimen comprise thesame 5′ portion. In some embodiments, the 5′ portion target-specificprimer can be configured to suppress primer dimers as described herein.

In some embodiments, first and second target-specific primers are usedin an amplification regimen that are substantially complementary to thesame strand of a target nucleic acid. In some embodiments, portions ofthe first and second target-specific primers that specifically anneal toa target sequence (e.g., a known target sequence) can comprise a totalof at least 20 unique bases of the known target nucleotide sequence,e.g. 20 or more unique bases, 25 or more unique bases, 30 or more uniquebases, 35 or more unique bases, 40 or more unique bases, or 50 or moreunique bases. In some embodiments, portions of first and secondtarget-specific primers that specifically anneal to a target sequence(e.g., a known target sequence) can comprise a total of at least 30unique bases of the known target nucleotide sequence.

As used herein, the term “first tail primer” refers to a nucleic acidmolecule comprising a nucleic acid sequence identical to the tailportion of tailed primer.

As used herein, the term “second tail primer” refers to a nucleic acidmolecule comprising a nucleic acid sequence identical to a portion of afirst sequencing primer, adapter, index primer, etc. and is optionallynested with respect to a first tailed primer. In some embodiments, thesecond tail primer sits outside of the first tail primer to facilitateaddition of appropriate index tags, adapters (e.g., for use in asequencing platform), etc. In some embodiments, a second tailed primeris identical to a sequencing primer. In some embodiments, a secondtailed primer is complementary to a sequencing primer.

In some embodiments, a second tail primer is nested with respect to afirst tail primer. In some embodiments, a second tail primer is notnested with respect to a first tail primer. In some embodiments, tailprimers of an amplification regimen are nested with respect to oneanother by at least 3 nucleotides, e.g. by 3 nucleotides, by 4nucleotides, by 5 nucleotides, by 6 nucleotides, by 7 nucleotides, by 8nucleotides, by 9 nucleotides, by 10 nucleotides or more.

In some embodiments, a first tail primer comprises a nucleic acidsequence identical to or complementary to the extension product of step(b) strand which is not comprised by the second tail primer and which islocated closer to the 5′ end of the tailed random primer than any of thesequence identical to or complementary to the second tail primer. Thus,in some embodiments, a second tail primer sits outside of a region addedby a random tail primer (5′ end), e.g., within the 5′ tail added by thefirst tail primers.

In some embodiments, a first tail primer can comprise a nucleic acidsequence identical to or complementary to a stretch (e.g., of about 20nucleotides) of the 5′-most nucleotides of a tailed random primer, and asecond tail primer can comprise a nucleic acid sequence identical to orcomplementary to about 30 bases of a tailed random primer, with a 5′nucleotides that is at least 3 nucleotides 3′ of the 5′ terminus of thetailed random primer.

In some embodiments, use of nested tail primers minimizes or eliminatesthe production of final amplicons that are amplifiable (e.g. duringbridge PCR or emulsion PCR) but cannot be sequenced, a situation thatcan arise during hemi-nested methods. In some embodiments, hemi-nestedapproaches using a primer identical to a sequencing primer can result inthe carry-over of undesired amplification products from a first PCR stepto a second PCR step and may yield artificial sequencing reads. In someembodiments, the use of two tail primers, as described herein canreduce, and in some embodiments eliminate, these problems.

In some embodiments, in a first PCR amplification cycle of a firstamplification step, a first target-specific primer can specificallyanneal to a template strand of any nucleic acid comprising the knowntarget nucleotide sequence. In some embodiments, depending upon theorientation with which the first target-specific primer was designed,sequence upstream or downstream of the known target nucleotide sequence,and complementary to the template strand will be synthesized. In someembodiments, in which an extension product is formed that comprises thehybridization sequence with which the first target-specific primer formscomplementary base pairs, adouble-stranded amplification product can beformed that comprises the first target-specific primer (and the sequencecomplementary thereto), the target nucleotide sequence downstream of thefirst target-specific primer (and the sequence complementary thereto),and the tailed random primer sequence (and the sequence complementarythereto). In such embodiments, in subsequent PCR amplification cycles,both the first target-specific primer and the first tail primer arecapable of specifically annealing to appropriate strands of theamplification product and the sequence between the known nucleotidetarget sequence and the tailed random primer can be amplified.

In some embodiments, of methods described herein, a portion of anamplified product (an amplicon) is amplified in further rounds ofamplification. In some embodiments, the further rounds of amplificationmay involve PCR amplification cycles performed using a secondtarget-specific primer and a first sequencing primer or a second tailprimer. In some embodiments, a PCR amplification cycles may involve theuse of PCR parameters identical to, or which differ from, those of oneor more other (e.g., prior) of PCR amplification cycles. In someembodiments, PCR amplification regimens can have the same or differentannealing temperatures or the same or different extension step timelengths.

In some embodiments, methods described herein allow for determining thenucleotide sequence contiguous to a known target nucleotide sequence oneither or both flanking regions of the known target nucleotide sequence.Regardless of whether the target nucleic acid normally exists as asingle-stranded or double-stranded nucleic acid, sequence informationmay be represented in a single-stranded format (Strand A), from 5′ to3′. In some embodiments, if the sequence 5′ to a known target nucleotidesequence of Strand A is to be determined, gene-specific primers can becomplementary to (anneal to) Strand A. If the sequence 3′ to a knowntarget nucleotide sequence of Strand A is to be determined, thegene-specific primers can be identical to Strand A, such that they willanneal to the complementary strand of a double-stranded target nucleicacid.

In some embodiments, methods described herein, relating to the use of afirst and second gene-specific primer can result in assays with asuperior on-target rate, e.g. 70-90%. In some embodiments, the assaysand methods described herein can have a target specificity rate of atleast 85%.

In some embodiments, primers disclosed herein (e.g., target-specificprimers, tail primers) are designed such that they will specificallyanneal to their complementary sequences at an annealing temperature offrom about 61 to 72° C., e.g. from about 61 to 69° C., from about 63 to69° C., from about 63 to 67° C., from about 64 to 66° C. In someembodiments, primers disclosed herein are designed such that they willspecifically anneal to their complementary sequences at an annealingtemperature of less than 72° C. In some embodiments, primers disclosedherein are designed such that they will specifically anneal to theircomplementary sequences at an annealing temperature of less than 70° C.In some embodiments, primers disclosed herein are designed such thatthey will specifically anneal to their complementary sequences at anannealing temperature of less than 68° C. In some embodiments, primersdisclosed herein are designed such that they will specifically anneal totheir complementary sequences at an annealing temperature of about 65°C.

In some embodiments, portions of the target-specific primers thatspecifically anneal to the known target nucleotide sequence will annealspecifically at a temperature of about 61 to 72° C., e.g. from about 61to 69° C., from about 63 to 69° C., from about 63 to 67° C., from about64 to 66° C. In some embodiments, portions of the target-specificprimers that specifically anneal to the known target nucleotide sequencewill anneal specifically at a temperature of about 65° C. in a PCRbuffer.

In some embodiments, primers described herein do not comprise modifiedbases (e.g. the primers can not comprise a blocking 3′ amine). However,in some embodiments, primers described herein do comprise modified ornon-naturally occurring bases. In some embodiments, primers may bemodified with a label capable of providing a detectable signal, eitherdirectly or indirectly. Non-limiting examples of such labels includeradioisotopes, fluorescent molecules, biotin, and others. In someembodiments, primers disclosed herein may include contain a biotinlinker or other suitable linker (e.g., for conjugating the primer to asupport). In some embodiments, primer may contain a target sequence ofan endonucleases such that cleavage with the appropriate enzyme. Inother embodiments, the 5′ end of a primer may include a sequence that iscomplementary with a nucleic acid bound to a bead or other support,e.g., a flow cell substrate. Primers may or may not comprise modifiedinternucleoside linkages.

In some embodiments, of methods described herein, nucleic acids (e.g.,amplified nucleic acids, extension products, target nucleic acids) canbe sequenced. In some embodiments, sequencing can be performed by anext-generation sequencing method. As used herein “next-generationsequencing” refers to oligonucleotide sequencing technologies that havethe capacity to sequence oligonucleotides at speeds above those possiblewith conventional sequencing methods (e.g. Sanger sequencing), due toperforming and reading out thousands to millions of sequencing reactionsin parallel. Non-limiting examples of next-generation sequencingmethods/platforms include Massively Parallel Signature Sequencing (LynxTherapeutics); 454 pyro-sequencing (454 Life Sciences/RocheDiagnostics); solid-phase, reversible dye-terminator sequencing(Solexa/Illumina): SOLiD technology (Applied Biosystems); Ionsemiconductor sequencing (ION Torrent); DNA nanoball sequencing(Complete Genomics); and technologies available from PacificBiosciences, Intelligen Bio-systems, Oxford Nanopore Technologies, andHelicos Biosciences. In some embodiments, the sequencing primers cancomprise portions compatible with the selected next-generationsequencing method. Next-generation sequencing technologies and theconstraints and design parameters of associated sequencing primers arewell known in the art (see, e.g. Shendure, et al., “Next-generation DNAsequencing,” Nature, 2008, vol. 26, No. 10, 1135-1145; Mardis, “Theimpact of next-generation sequencing technology on genetics,” Trends inGenetics, 2007, vol. 24, No. 3, pp. 133-141; Su, et al.,“Next-generation sequencing and its applications in moleculardiagnostics” Expert Rev Mol Diagn, 2011, 11(3):333-43; Zhang et al.,“The impact of next-generation sequencing on genomics”, J GenetGenomics, 2011, 38(3):95-109; (Nyren, P. et al. Anal Biochem 208: 17175(1993); Bentley, D. R. Curr Opin Genet Dev 16:545-52 (2006); Strausberg,R. L., et al. Drug Disc Today 13:569-77 (2008); U.S. Pat. Nos.7,282,337; 7,279,563; 7,226,720; 7,220,549; 7,169,560; 6,818,395;6,911,345; US Pub. Nos. 2006/0252077; 2007/0070349; and 20070070349;which are incorporated by reference herein in their entireties).

In some embodiments, the sequencing step involve the use of a first andsecond sequencing primers. In some embodiments, the first and secondsequencing primers are selected to be compatible with a next-generationsequencing method as described herein.

Methods of aligning sequencing reads to known sequence databases ofgenomic and/or cDNA sequences are well known in the art and software iscommercially available for this process. In some embodiments, reads(less the sequencing primer nucleotide sequence) which do not map, intheir entirety, to wild-type sequence databases can be genomicrearrangements or large indel mutations. In some embodiments, reads(less the sequencing primer nucleotide sequence) comprising sequenceswhich map to multiple locations in the genome can be genomicrearrangements.

In some embodiments, primers may contain additional sequences such as anidentifier sequence (e.g., a barcode, an index), sequencing primerhybridization sequences (e.g., Rd1), and adapter sequences. In someembodiments the adapter sequences are sequences used with a nextgeneration sequencing system. In some embodiments, the adapter sequencesare P5 and P7 sequences for Illumina-based sequencing technology. Insome embodiments, the adapter sequence are P1 and A compatible with IonTorrent sequencing technology.

In some embodiments, as used herein, a “barcode,” “molecular barcode,”“molecular barcode tag” and “index” may be used interchangeably.generally referring to a nucleotide sequence of a nucleic acid that isuseful as an identifier, such as, for example, a source identifier,location identifier, date or time identifier (e.g., date or time ofsampling or processing), or other identifier of the nucleic acid. Insome embodiments, such barcode or index sequences are useful foridentifying different aspects of a nucleic acid that is present in apopulation of nucleic acids. In some embodiments, barcode or indexsequences may provide a source or location identifier for a targetnucleic acid. For example, a barcode or index sequence may serve toidentify a patient from whom a nucleic acid is obtained. In someembodiments, barcode or index sequences enable sequencing of multipledifferent samples on a single reaction (e.g., performed in a single flowcell). In some embodiments, an index sequence can be used to orientate asequence imager for purposes of detecting individual sequencingreactions. In some embodiments, a barcode or index sequence may be 2 to25 nucleotides in length, 2 to 15 nucleotides in length, 2 to 10nucleotides in length, 2 to 6 nucleotides in length. In someembodiments, a barcode or index comprise at least 2, 3, 4, 5, 6, 7 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or at least25 nucleotides.

In some embodiments, when a population of tailed random primers is usedin accordance with methods described herein, multiple distinguishableamplification products can be present after amplification. In someembodiments, because tailed random primers hybridize at variouspositions throughout nucleic acid molecules of a sample, a set oftarget-specific primers can hybridize (and amplify) the extensionproducts created by more than 1 hybridization event, e.g. one tailedrandom primer may hybridize at a first distance (e.g., 100 nucleotides)from a target-specific primer hybridization site, and another tailedrandom primer can hybridize at a second distance (e.g., 200 nucleotides)from a target-specific primer hybridization site, thereby resulting intwo amplification products (e.g., a ˜100 bp amplification product and a˜200 bp amplification product). In some embodiments, these multipleamplification products can each be sequenced in. In some embodiments,sequencing of these multiple amplification products is advantageousbecause it provides multiple overlapping sequence reads that can becompare with one another to detect sequence errors introduced duringamplification or sequencing processes. In some embodiments, individualamplification products can be aligned and where they differ in thesequence present at a particular base, an artifact or error of PCRand/or sequencing may be present.

In some embodiments, target nucleic acids and/or amplification productsthereof can be isolated from enzymes, primers, or buffer componentsbefore and/or after any of appropriate step of a method. Any suitablemethods for isolating nucleic acids may be used. In some embodiments,the isolation can comprise Solid Phase Reversible Immobilization (SPRI)cleanup. Methods for SPRI cleanup are well known in the art and kits arecommercially available, e.g. Agencourt AMPure XP-PCR Purification (CatNo. A63880, Beckman Coulter; Brea, Calif.). In some embodiments, enzymescan be inactivated by heat treatment.

In some embodiments, unhybridized primers can be removed from a nucleicacid preparation using appropriate methods (e.g., purification,digestion, etc.). In some embodiments, a nuclease (e.g., exonuclease I)is used to remove primer from a preparation. In some embodiments, suchnucleases are heat inactivated subsequent to primer digestion. Once thenucleases are inactivated a further set of primers may be added togetherwith other appropriate components (e.g., enzymes, buffers) to perform afurther amplification reaction.

In some embodiments, a target nucleic acid genomic DNA or a portionthereof. In some embodiments, a target nucleic acid can be ribonucleicacid (RNA), e.g. mRNA, or a portion thereof. In some embodiments, atarget nucleic acid can be a cDNA or a portion thereof.

Many of the sequencing methods suitable for use in methods describedherein provide sequencing runs with optimal read lengths of tens tohundreds of nucleotide bases (e.g. Ion Torrent technology can produceread lengths of 200-400 bp). Target nucleic acids may or may not besubstantially longer than this optimal read length. In some embodiments,in order for an amplified nucleic acid portion to be of a suitablelength for use in a particular sequencing technology, the averagedistance between the known target nucleotide sequence and an end of thetarget nucleic acid to which a tailed random primer is hybridizableshould be as close to the optimal read length of the selected technologyas possible. In some embodiments, if the optimal read-length of a givensequencing technology is 200 bp, then the nucleic acid moleculesamplified in accordance with methods described herein should have anaverage length of about 800 bp, about 700 bp, about 600 bp, about 500bp, about 400 bp, about 300 bp, about 200 bp or less.

Nucleic acids used herein (e.g., prior to sequencing) can be sheared,e.g. mechanically or enzymatically sheared, to generate fragments of anydesired size. Non-limiting examples of mechanical shearing processesinclude sonication, nebulization, and AFA™ shearing technology availablefrom Covaris (Woburn, Mass.). In some embodiments, a nucleic acid can bemechanically sheared by sonication.

In some embodiments, a target nucleic acid is not sheared or digested.In some embodiments, nucleic acid products of preparative steps (e.g.,extension products, amplification products) are not sheared orenzymatically digested.

In some embodiments, when a target nucleic acid an RNA, the sample canbe subjected to a reverse transcriptase regimen to generate DNA templateand the DNA template can then be sheared. In some embodiments, targetRNA can be sheared before performing a reverse transcriptase regimen. Insome embodiments, a sample comprising target RNA can be used in methodsdescribed herein using total nucleic acids extracted from either freshor degraded specimens; without the need of genomic DNA removal for cDNAsequencing; without the need of ribosomal RNA depletion for cDNAsequencing; without the need of mechanical or enzymatic shearing in anyof the steps; by subjecting the RNA for double-stranded cDNA synthesisusing random hexamers.

In some embodiments, a known target nucleic acid can contain a fusionsequence resulting from a gene rearrangement. In some embodiments,methods described herein are suited for determining the presence and/oridentity of a gene rearrangement. In some embodiments, identity of oneportion of a gene rearrangement is previously known (e.g., the portionof a gene rearrangement that is to be targeted by the gene-specificprimers) and the sequence of the other portion may be determined usingmethods disclosed herein. In some embodiments, a gene rearrangement caninvolves an oncogene. In some embodiments, a gene rearrangement cancomprise a fusion oncogene.

In some embodiments, a target nucleic acid is present in or obtainedfrom an appropriate sample (e.g., a food sample, environmental sample,biological sample e.g., blood sample, etc.). In some embodiments, the isa biological sample obtained from a subject. In some embodiments asample can be a diagnostic sample obtained from a subject. In someembodiments, a sample can further comprise proteins, cells, fluids,biological fluids, preservatives, and/or other substances. By way ofnon-limiting example, a sample can be a cheek swab, blood, serum,plasma, sputum, cerebrospinal fluid, urine, tears, alveolar isolates,pleural fluid, pericardial fluid, cyst fluid, tumor tissue, tissue, abiopsy, saliva, an aspirate, or combinations thereof. In someembodiments, a sample can be obtained by resection or biopsy.

In some embodiments, the sample can be obtained from a subject in needof treatment for a disease associated with a genetic alteration, e.g.cancer or a herediatary disease. In some embodiments, a known targetsequence is present in a disease-associated gene.

In some embodiments, a sample is obtained from a subject in need oftreatment for cancer. In some embodiments, the sample comprises apopulation of tumor cells, e.g. at least one tumor cell. In someembodiments, the sample comprises a tumor biopsy, including but notlimited to, untreated biopsy tissue or treated biopsy tissue (e.g.formalin-fixed and/or paraffin-embedded biopsy tissue).

In some embodiments, the sample is freshly collected. In someembodiments, the sample is stored prior to being used in methods andcompositions described herein. In some embodiments, the sample is anuntreated sample. As used herein, “untreated sample” refers to abiological sample that has not had any prior sample pre-treatment exceptfor dilution and/or suspension in a solution. In some embodiments, asample is obtained from a subject and preserved or processed prior tobeing utilized in methods and compositions described herein. By way ofnon-limiting example, a sample can be embedded in paraffin wax,refrigerated, or frozen. A frozen sample can be thawed beforedetermining the presence of a nucleic acid according to methods andcompositions described herein. In some embodiments, the sample can be aprocessed or treated sample. Exemplary methods for treating orprocessing a sample include, but are not limited to, centrifugation,filtration, sonication, homogenization, heating, freezing and thawing,contacting with a preservative (e.g. anti-coagulant or nucleaseinhibitor) and any combination thereof. In some embodiments, a samplecan be treated with a chemical and/or biological reagent. Chemicaland/or biological reagents can be employed to protect and/or maintainthe stability of the sample or nucleic acid comprised by the sampleduring processing and/or storage. In addition, or alternatively,chemical and/or biological reagents can be employed to release nucleicacids from other components of the sample. By way of non-limitingexample, a blood sample can be treated with an anti-coagulant prior tobeing utilized in methods and compositions described herein. Suitablemethods and processes for processing, preservation, or treatment ofsamples for nucleic acid analysis may be used in the method disclosedherein. In some embodiments, a sample can be a clarified fluid sample,for example, by centrifugation. In some embodiments, a sample can beclarified by low-speed centrifugation (e.g. 3,000×g or less) andcollection of the supernatant comprising the clarified fluid sample.

In some embodiments, a nucleic acid present in a sample can be isolated,enriched, or purified prior to being utilized in methods andcompositions described herein. Suitable methods of isolating, enriching,or purifying nucleic acids from a sample may be used. For example, kitsfor isolation of genomic DNA from various sample types are commerciallyavailable (e.g. Catalog Nos. 51104, 51304, 56504, and 56404; Qiagen;Germantown, Md.). In some embodiments, methods described herein relateto methods of enriching for target nucleic acids, e.g., prior to asequencing of the target nucleic acids. In some embodiments, a sequenceof one end of the target nucleic acid to be enriched is not known priorto sequencing. In some embodiments, methods described herein relate tomethods of enriching specific nucleotide sequences prior to determiningthe nucleotide sequence using a next-generation sequencing technology.In some embodiments, methods of enriching specific nucleotide sequencesdo not comprise hybridization enrichment.

Methods described herein can be employed in a multiplex format. Inembodiments of methods described herein, multiplex applications caninclude determining the nucleotide sequence contiguous to one or moreknown target nucleotide sequences. As used herein, “multiplexamplification” refers to a process involve simultaneous amplification ofmore than one target nucleic acid in one reaction vessel. In someembodiments, methods involve subsequent determination of the sequence ofthe multiplex amplification products using one or more sets of primers.Multiplex can refer to the detection of between about 2-1,000 differenttarget sequences in a single reaction. As used herein, multiplex refersto the detection of any range between 2-1,000, e.g., between 5-500,25-1000, or 10-100 different target sequences in a single reaction, etc.The term “multiplex” as applied to PCR implies that there are primersspecific for at least two different target sequences in the same PCRreaction.

In some embodiments, target nucleic acids in a sample, or separateportions of a sample, can be amplified with a plurality of primers(e.g., a plurality of first and second target-specific primers). In someembodiments, the plurality of primers (e.g., a plurality of first andsecond target-specific primers) can be present in a single reactionmixture, e.g. multiple amplification products can be produced in thesame reaction mixture. In some embodiments, the plurality of primers(e.g., a plurality of sets of first and second target-specific primers)can specifically anneal to known target sequences comprised by separategenes. In some embodiments, at least two sets of primers (e.g., at leasttwo sets of first and second target-specific primers) can specificallyanneal to different portions of a known target sequence. In someembodiments, at least two sets of primers (e.g., at least two sets offirst and second target-specific primers) can specifically anneal todifferent portions of a known target sequence comprised by a singlegene. In some embodiments, at least two sets of primers (e.g., at leasttwo sets of first and second target-specific primers) can specificallyanneal to different exons of a gene comprising a known target sequence.In some embodiments, the plurality of primers (e.g., firsttarget-specific primers) can comprise identical 5′ tag sequenceportions.

In embodiments of methods described herein, multiplex applications caninclude determining the nucleotide sequence contiguous to one or moreknown target nucleotide sequences in multiple samples in one sequencingreaction or sequencing run. In some embodiments, multiple samples can beof different origins, e.g. from different tissues and/or differentsubjects. In such embodiments, primers (e.g., tailed random primers) canfurther comprise a barcode portion. In some embodiments, a primer (e.g.,a tailed random primer) with a unique barcode portion can be added toeach sample and ligated to the nucleic acids therein; the samples cansubsequently be pooled. In such embodiments, each resulting sequencingread of an amplification product will comprise a barcode that identifiesthe sample containing the template nucleic acid from which theamplification product is derived.

In some embodiments of methods described herein, a determination of thesequence contiguous to a known oligonucleotide target sequence canprovide information relevant to treatment of disease. Thus, in someembodiments, methods disclosed herein can be used to aid in treatingdisease. In some embodiments, a sample can be from a subject in need oftreatment for a disease associated with a genetic alteration. In someembodiments, a known target sequence a sequence of a disease-associatedgene, e.g. an oncogene. In some embodiments, a sequence contiguous to aknown oligonucleotide target sequence and/or the known oligonucleotidetarget sequence can comprise a mutation or genetic abnormality which isdisease-associated, e.g. a SNP, an insertion, a deletion, and/or a generearrangement. In some embodiments, a sequence contiguous to a knowntarget sequence and/or a known target sequence present in a samplecomprised sequence of a gene rearrangement product. In some embodiments,a gene rearrangement can be an oncogene, e.g. a fusion oncogene.

Certain treatments for cancer are particularly effective against tumorscomprising certain oncogenes, e.g. a treatment agent which targets theaction or expression of a given fusion oncogene can be effective againsttumors comprising that fusion oncogene but not against tumors lackingthe fusion oncogene. Methods described herein can facilitate adetermination of specific sequences that reveal oncogene status (e.g.mutations, SNPs, and/or rearrangements). In some embodiments, methodsdescribed herein can further allow the determination of specificsequences when the sequence of a flanking region is known, e.g. methodsdescribed herein can determine the presence and identity of generearrangements involving known genes (e.g., oncogenes) in which theprecise location and/or rearrangement partner are not known beforemethods described herein are performed.

In some embodiments, technology described herein relates to a method oftreating cancer. Accordingly, in some embodiments, methods providedherein may involve detecting, in a tumor sample obtained from a subjectin need of treatment for cancer, the presence of one or more oncogenerearrangements; and administering a cancer treatment which is effectiveagainst tumors having any of the detected oncogene rearrangements. Insome embodiments, technology described herein relates to a method ofdetermining if a subject in need of treatment for cancer will beresponsive to a given treatment. Accordingly, in some embodiments,methods provided herein may involve detecting, in a tumor sampleobtained from a subject, the presence of an oncogene rearrangement, inwhich the subject is determined to be responsive to a treatmenttargeting an oncogene rearrangement product if the presence of theoncogene rearrangement is detected.

In some embodiments, a subject is in need of treatment for lung cancer.In some embodiments, e.g. when the sample is obtained from a subject inneed of treatment for lung cancer, the known target sequence cancomprise a sequence from a gene selected from the group of ALK, ROS1,and RET. Accordingly, in some embodiments, gene rearrangements result infusions involving the ALK, ROS1, or RET. Non-limiting examples of genearrangements involving ALK, ROS1, or RET are described in, e.g., Soda etal. Nature 2007 448561-6: Rikova et al. Cell 2007 131:1190-1203; Kohnoet al. Nature Medicine 2012 18:375-7; Takouchi et al. Nature Medicine2012 18:378-81; which are incorporated by reference herein in theirentireties. However, it should be appreciated that the precise locationof a gene rearrangement, and the identity of the second gene involved inthe rearrangement may not be known in advance. Accordingly, in methodsdescribed herein, the presence and identity of such rearrangements canbe detected without having to know the location of the rearrangement orthe identity of the second gene involved in the gene rearrangement.

In some embodiments, the known target sequence can comprise sequencefrom a gene selected from the group of: ALK, ROS1, and RET.

In some embodiments, the presence of a gene rearrangement of ALK in asample obtained from a tumor in a subject can indicate that the tumor issusceptible to treatment with a treatment selected from the groupconsisting of: an ALK inhibitor; crizotinib (PF-02341066); AP26113;LDK378; 3-39; AF802; IPI-504; ASP3026; AP-26113; X-396; GSK-1838705A;CH5424802; diamino and aminopyrimidine inhibitors of ALK kinase activitysuch as NVP-TAE684 and PF-02341066 (see, e.g. Galkin et al, Proc NatlAcad Sci USA, 2007, 104:270-275; Zou et al. Cancer Res, 2007,67:4408-4417; Hallberg and Palmer F1000 Med Reports 2011 3:21; andSakamoto et al. Cancer Cell 2011 19:679-690) and molecules disclosed inWO 04/079326. All of the foregoing references are incorporated byreference herein in their entireties. An ALK inhibitor can include anyagent that reduces the expression and/or kinase activity of ALK or aportion thereof, including, e.g. oligonucleotides, small molecules,and/or peptides that reduce the expression and/or activity of ALK or aportion thereof. As used herein “anaplastic lymphoma kinase” or “ALK”refers to a transmembrane tyROS line kinase typically involved inneuronal regulation in the wildtype form. The nucleotide sequence of theALK gene and mRNA are known for a number of species, including human(e.g. SEQ ID NO: 2 (mRNA), NCBI Gene ID: 238).

In some embodiments, the presence of a gene rearrangement of ROS1 in asample obtained from a tumor in a subject can indicate that the tumor issusceptible to treatment with a treatment selected from the groupconsisting of: a ROS1 inhibitor and an ALK inhibitor as described hereinabove (e.g. crizotinib). A ROS1 inhibitor can include any agent thatreduces the expression and/or kinase activity of ROS1 or a portionthereof, including, e.g. oligonucleotides, small molecules, and/orpeptides that reduce the expression and/or activity of ROS1 or a portionthereof. As used herein “c-ros oncogene 1” or “ROS1” (also referred toin the art as ros-1) refers to a transmembrane tyrosine kinase of thesevenless subfamily and which interacts with PTPN6. Nucleotide sequencesof the ROS1 gene and mRNA are known for a number of species, includinghuman (e.g. SEQ ID NO: 1 (mRNA), NCBI Gene ID: 238).

In some embodiments, the presence of a gene rearrangement of RET in asample obtained from a tumor in a subject can indicate that the tumor issusceptible to treatment with a treatment selected from the groupconsisting of: a RET inhibitor; DP-2490, DP-3636, SU5416; BAY 43-9006,BAY 73-4506 (regorafenib), ZD6474, NVP-AST487, sorafenib, RPI-1, XL184,vandetanib, sunitinib, imatinib, pazopanib, axitinib, motesanib,gefitinib, and withaferin A (see, e.g. Samadi et al. Surgery 2010148:1228-36; Cuccuru et al. JNCI 2004 13:1006-1014; Akeno-Stuart et al.Cancer Research 2007 67:6956; Grazma et al. J Clin Oncol 2010 28:15s5559; Mologni e tal. J Mol Endocrinol 2006 37:199-212; Calmomagno et al.Journal NCI 2006 98:326-334; Mologni. Curr Med Chem 2011 18:162-175 andthe compounds disclosed in WO 06/034833; US Patent Publication2011/0201598 and U.S. Pat. No. 8,067,434). All of the foregoingreferences are incorporated by reference herein in their entireties. ARET inhibitor can include any agent that reduces the expression and/orkinase activity of RET or a portion thereof, including, e.g.oligonucleotides, small molecules, and/or peptides that reduce theexpression and/or activity of RET or a portion thereof. As used herein“rearranged during transfection” or “RET” refers to a receptor tyrosinekinase of the cadherein superfamily which is involved in neural crestdevelopment and recognizes glial cell line-derived neurotrophic factorfamily signaling molecules. Nucleotide sequences of the RET gene andmRNA are known for a number of species, including human (e.g. SEQ IDNOs: 3-4 (mRNA), NCBI Gene ID: 5979).

Further non-limiting examples of applications of methods describedherein include detection of hematological malignancy markers and panelsthereof (e.g. including those to detect genomic rearrangements inlymphomas and leukemias), detection of sarcoma-related genomicrearrangements and panels thereof; and detection of IGH/TCR generearrangements and panels thereof for lymphoma testing.

In some embodiments, methods described herein relate to treating asubject having or diagnosed as having, e.g. cancer with a treatment forcancer. Subjects having cancer can be identified by a physician usingcurrent methods of diagnosing cancer. For example, symptoms and/orcomplications of lung cancer which characterize these conditions and aidin diagnosis are well known in the art and include but are not limitedto, weak breathing, swollen lymph nodes above the collarbone, abnormalsounds in the lungs, dullness when the chest is tapped, and chest pain.Tests that may aid in a diagnosis of, e.g. lung cancer include, but arenot limited to, x-rays, blood tests for high levels of certainsubstances (e.g. calcium), CT scans, and tumor biopsy. A family historyof lung cancer, or exposure to risk factors for lung cancer (e.g.smoking or exposure to smoke and/or air pollution) can also aid indetermining if a subject is likely to have lung cancer or in making adiagnosis of lung cancer.

Cancer can include, but is not limited to, carcinoma, includingadenocarcinoma, lymphoma, blastoma, melanoma, sarcoma, leukemia,squamous cell cancer, small-cell lung cancer, non-small cell lungcancer, gastrointestinal cancer, Hodgkin's and non Hodgkin's lymphoma,pancreatic cancer, glioblastoma, basal cell carcinoma, biliary tractcancer, bladder cancer, brain cancer including glioblastomas andmedulloblastomas; breast cancer, cervical cancer, choriocarcinoma; coloncancer, colorectal cancer, endometrial carcinoma, endometrial cancer;esophageal cancer, gastric cancer; various types of head and neckcancers, intraepithelial neoplasms including Bowen's disease and Paget'sdisease; hematological neoplasms including acute lymphocytic andmyelogenous leukemia; Kaposi's sarcoma, hairy cell leukemia; chromicmyelogenous leukemia, AIDS-associated leukemias and adult T-cellleukemia lymphoma; kidney cancer such as renal cell carcinoma, T-cellacute lymphoblastic leukemia/lymphoma, lymphomas including Hodgkin'sdisease and lymphocytic lymphomas; liver cancer such as hepaticcarcinoma and hepatoma, Merkel cell carcinoma, melanoma, multiplemyeloma; neuroblastomas; oral cancer including squamous cell carcinoma;ovarian cancer including those arising from epithelial cells, sarcomasincluding leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibROS1arcoma,and osteosarcoma; pancreatic cancer; skin cancer including melanoma,stromal cells, germ cells and mesenchymal cells; pROS1tate cancer,rectal cancer; vulval cancer, renal cancer including adenocarcinoma;testicular cancer including germinal tumors such as seminoma,non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germcell tumors; thyroid cancer including thyroid adenocarcinoma andmedullar carcinoma; esophageal cancer, salivary gland carcinoma, andWilms' tumors. In some embodiments, the cancer can be lung cancer.

In some embodiments, methods described herein comprise administering aneffective amount of compositions described herein, e.g. a treatment forcancer to a subject in order to alleviate a symptom of a cancer. As usedherein, “alleviating a symptom of a cancer” is ameliorating anycondition or symptom associated with the cancer. As compared with anequivalent untreated control, such reduction is by at least 5%, 10%,20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by anystandard technique. A variety of means for administering thecompositions described herein to subjects are known to those of skill inthe art. Such methods can include, but are not limited to oral,parenteral, intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol), pulmonary, cutaneous, topical, injection, orintratumoral administration. Administration can be local or systemic.The term “effective amount” as used herein refers to the amount of atreatment needed to alleviate at least one or more symptom of thedisease or disorder, and relates to a sufficient amount ofpharmacological composition to provide the desired effect. The term“therapeutically effective amount” therefore refers to an amount that issufficient to effect a particular anti-cancer effect when administeredto a typical subject. An effective amount as used herein, in variouscontexts, would also include an amount sufficient to delay thedevelopment of a symptom of the disease, alter the course of a symptomdisease (for example but not limited to, slowing the progression of asymptom of the disease), or reverse a symptom of the disease. Thus, itis not generally practicable to specify an exact “effective amount”.However, for any given case, an appropriate “effective amount” can bedetermined by one of ordinary skill in the art using only routineexperimentation. The effects of any particular dosage can be monitoredby a suitable bioassay. The dosage can be determined by a physician andadjusted, as appropriate, to suit observed effects of the treatment.

Non-limiting examples of a treatment for cancer can include radiationtherapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin,bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin,ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1 and calicheamicin omega1(see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above. In addition, methods of treatment canfurther include the use of radiation or radiation therapy. Further,methods of treatment can further include the use of surgical treatments.

In some embodiments, methods described herein can be applicable forresequencing, e.g. for confirming particularly relevant, low-quality,and/or complex sequences obtained by non-directed sequencing of a largeamount of nucleic acids. By way of non-limiting examples, methodsdescribed herein can allow the directed and/or targeted resequencing oftargeted disease gene panels (e.g. 10-100 genes), resequencing toconfirm variants obtained in large scale sequencing projects, wholeexome resequencing, and/or targeted resequencing for detection of singlenucleotide variants, multiple nucleotide variants, insertions,deletions, copy number changes, and methylation status.

In some embodiments, methods described herein can allow microbiotasequencing, ancient sample sequencing, and/or new variant virusgenotyping.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein generally to mean a decrease by a statistically significantamount. However, for avoidance of doubt, “reduced”, “reduction”,“decrease”, or “inhibit” means a decrease by at least 10% as compared toa reference level, for example a decrease by at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% decrease (e.g. absentlevel or non-detectable level as compared to a reference level), or anydecrease between 10-100% as compared to a reference level. In thecontext of a marker or symptom is meant a statistically significantdecrease in such level. The decrease can be, for example, at least 10%,at least 20%, at least 30%, at least 40% or more, and is preferably downto a level accepted as within the range of normal for an individualwithout such disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to generally mean an increase by a statically significant amount;for the avoidance of doubt, the terms “increased”, “increase”,“enhance”, or “activate” mean an increase of at least 10% as compared toa reference level, for example an increase of at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% increase or any increasebetween 10-100% as compared to a reference level, or at least about a2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of, e.g.lung cancer. A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g. cancer) or one or more complications related to such a condition,and optionally, have already undergone treatment for the condition orthe one or more complications related to the condition. Alternatively, asubject can also be one who has not been previously diagnosed as havingthe condition (e.g. cancer) or one or more complications related to thecondition. For example, a subject can be one who exhibits one or morerisk factors for the condition or one or more complications related tothe condition or a subject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, a “disease associated with a genetic alteration” refersto any disease which is caused by, at least in part, by an alteration inthe genetic material of the subject as compared to a healthy wildtypesubject, e.g. a deletion, an insertion, a SNP, a gene rearrangement. Adisease can be caused by, at least in part, an alteration in the geneticmaterial of the subject if the alteration increases the risk of thesubject developing the disease, increases the subject's susceptibilityto a disease (including infectious diseases, or diseases with aninfectious component), causes the production of a disease-associatedmolecule, or causes cells to become diseased or abnormal (e.g. loss ofcell cycle regulation in cancer cells). Diseases can be associated withmultiple genetic alterations, e.g. cancers.

As used herein, the term “nucleic acid” refers to any molecule,preferably a polymeric molecule, incorporating units of ribonucleicacid, deoxyribonucleic acid or an analog thereof. The nucleic acid canbe either single-stranded or double-stranded. A single-stranded nucleicacid can be one strand nucleic acid of a denatured double-stranded DNA.Alternatively, it can be a single-stranded nucleic acid not derived fromany double-stranded DNA. In one aspect, the template nucleic acid isDNA. In another aspect, the template is RNA. Suitable nucleic acidmolecules are DNA, including genomic DNA or cDNA. Other suitable nucleicacid molecules are RNA, including mRNA.

The term “isolated” or “partially purified” as used herein refers, inthe case of a nucleic acid, to a nucleic acid separated from at leastone other component (e.g., nucleic acid or polypeptide) that is presentwith the nucleic acid as found in its natural source and/or that wouldbe present with the nucleic acid when expressed by a cell. A chemicallysynthesized nucleic acid or one synthesized using in vitrotranscription/translation is considered “isolated.”

As used herein, the term “complementary” refers to the ability ofnucleotides to form hydrogen-bonded base pairs. In some embodiment,complementary refers to hydrogen-bonded base pair formation preferencesbetween the nucleotide bases G, A, T, C and U, such that when two givenpolynucleotides or polynucleotide sequences anneal to each other, Apairs with T and G pairs with C in DNA, and G pairs with C and A pairswith U in RNA. As used herein, “substantially complementary” refers to anucleic acid molecule or portion thereof (e.g. a primer) having at least90% complementarity over the entire length of the molecule or portionthereof with a second nucleotide sequence, e.g. 90% complementary, 95%complementary, 98% complementary, 99% complementary, or 100%complementary. As used herein, “substantially identical” refers to anucleic acid molecule or portion thereof having at least 90% identityover the entire length of a the molecule or portion thereof with asecond nucleotide sequence, e.g. 90% identity, 95% identity, 98%identity, 99% identity, or 100% identity.

As used herein, “specific” when used in the context of a primer specificfor a target nucleic acid refers to a level of complementarity betweenthe primer and the target such that there exists an annealingtemperature at which the primer will anneal to and mediate amplificationof the target nucleic acid and will not anneal to or mediateamplification of non-target sequences present in a sample.

As used herein, “amplified product”, “amplification product”, or“amplicon” refers to oligonucleotides resulting from an amplificationreaction that are copies of a portion of a particular target nucleicacid template strand and/or its complementary sequence, which correspondin nucleotide sequence to the template nucleic acid sequence and/or itscomplementary sequence. An amplification product can further comprisesequence specific to the primers and which flanks sequence which is aportion of the target nucleic acid and/or its complement. An amplifiedproduct, as described herein will generally be double-stranded DNA,although reference can be made to individual strands thereof.

As used herein, a “portion” of a nucleic acid molecule refers tocontiguous set of nucleotides comprised by that molecule. A portion cancomprise all or only a subset of the nucleotides comprised by themolecule. A portion can be double-stranded or single-stranded.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. lung cancer. The term “treating” includes reducing oralleviating at least one adverse effect or symptom of a condition,disease or disorder associated with a condition. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of, or at leastslowing of, progress or worsening of symptoms compared to what would beexpected in the absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total), and/or decreased mortality, whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9).Definitions of common terms in molecular biology can also be found inBenjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009(ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols inProtein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2001); and Davis etal., Basic Methods in Molecular Biology, Elsevier Science Publishing,Inc., New York, USA (1995) which are all incorporated by referenceherein in their entireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, methodologies described in such publicationsthat might be used in connection with technology described herein. Thesepublications are provided solely for their disclosure prior to thefiling date of the present application. Nothing in this regard should beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention or for any otherreason. All statements as to the date or representation as to thecontents of these documents is based on the information available to theapplicants and does not constitute any admission as to the correctnessof the dates or contents of these documents.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if appropriate, to employ the compositions, functions and concepts ofthe above references and application to provide yet further embodimentsof the disclosure. These and other changes can be made to the disclosurein light of the detailed description. All such modifications areintended to be included within the scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

Technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

1. A method of determining the nucleotide sequence contiguous to a knowntarget nucleotide sequence, the method comprising;

-   -   (a) contacting a target nucleic acid molecule comprising the        known target nucleotide sequence with an initial target-specific        primer under hybridization conditions;    -   (b) performing a template-dependent extension reaction that is        primed by a hybridized initial target-specific primer and that        uses the target nucleic acid molecule as a template;    -   (c) contacting the product of step (b) with a population of        tailed random primers under hybridization conditions;    -   (d) performing a template-dependent extension reaction that is        primed by a hybridized tailed random primer and that uses the        portion of the target nucleic acid molecule downstream of the        site of hybridization as a template;    -   (e) amplifying a portion of the target nucleic acid molecule and        the tailed random primer sequence with a first tail primer and a        first target-specific primer;    -   (f) amplifying a portion of the amplicon resulting from step (e)        with a second tail primer and a second target-specific primer;    -   (g) sequencing the amplified portion from step (f) using a first        and second sequencing primer;

wherein the population of tailed random primers comprisessingle-stranded oligonucleotide molecules having a 5′ nucleic acidsequence identical to a first sequencing primer and a 3′ nucleic acidsequence comprising from about 6 to about 12 random nucleotides;

wherein the first target-specific primer comprises a nucleic acidsequence that can specifically anneal to the known target nucleotidesequence of the target nucleic acid at the annealing temperature;

wherein the second target-specific primer comprises a 3′ portioncomprising a nucleic acid sequence that can specifically anneal to aportion of the known target nucleotide sequence comprised by theamplicon resulting from step (e), and a 5′ portion comprising a nucleicacid sequence that is identical to a second sequencing primer and thesecond target-specific primer is nested with respect to the firsttarget-specific primer;

wherein the first tail primer comprises a nucleic acid sequenceidentical to the tailed random primer; and

wherein the second tail primer comprises a nucleic acid sequenceidentical to a portion of the first sequencing primer and is nested withrespect to the first tail primer.

2. A method of determining the nucleotide sequence contiguous to a knowntarget nucleotide sequence, the method comprising;

-   -   (a) contacting a target nucleic acid molecule comprising the        known target nucleotide sequence with a population of tailed        random primers under hybridization conditions;    -   (b) performing a template-dependent extension reaction that is        primed by a hybridized tailed random primer and that uses the        portion of the target nucleic acid molecule downstream of the        site of hybridization as a template;    -   (c) contacting the product of step (b) with an initial        target-specific primer under hybridization conditions;    -   (d) performing a template-dependent extension reaction that is        primed by a hybridized initial target-specific primer and that        uses the target nucleic acid molecule as a template;    -   (e) amplifying a portion of the target nucleic acid molecule and        the tailed random primer sequence with a first tail primer and a        first target-specific primer;    -   (f) amplifying a portion of the amplicon resulting from step (e)        with a second tail primer and a second target-specific primer;    -   (g) sequencing the amplified portion from step (f) using a first        and second sequencing primer;

wherein the population of tailed random primers comprisessingle-stranded oligonucleotide molecules having a 5′ nucleic acidsequence identical to a first sequencing primer and a 3′ nucleic acidsequence comprising from about 6 to about 12 random nucleotides;

wherein the first target-specific primer comprises a nucleic acidsequence that can specifically anneal to the known target nucleotidesequence of the target nucleic acid at the annealing temperature;

wherein the second target-specific primer comprises a 3′ portioncomprising a nucleic acid sequence that can specifically anneal to aportion of the known target nucleotide sequence comprised by theamplicon resulting from step (c), and a 5′ portion comprising a nucleicacid sequence that is identical to a second sequencing primer and thesecond target-specific primer is nested with respect to the firsttarget-specific primer;

wherein the first tail primer comprises a nucleic acid sequenceidentical to the tailed random primer; and

wherein the second tail primer comprises a nucleic acid sequenceidentical to a portion of the first sequencing primer and is nested withrespect to the first tail primer.

3. The method of any of paragraphs 1-2, further comprising a step ofcontacting the sample and products with RNase after extension of theinitial target-specific primer.

4. The method of any of paragraphs 1-3, wherein the tailed random primercan form a hair-pin loop structure.

5. The method of any of paragraphs 1-4, wherein the initialtarget-specific primer and the first target-specific primer areidentical.

6. The method of any of paragraphs 1-5, wherein the tailed random primerfurther comprises a barcode portion comprising 6-12 random nucleotidesbetween the 5′ nucleic acid sequence identical to a first sequencingprimer and the 3′ nucleic acid sequence comprising 6-12 randomnucleotides. 7. A method of determining the nucleotide sequencecontiguous to a known target nucleotide sequence, the method comprising;

-   -   (a) contacting a target nucleic acid molecule comprising the        known target nucleotide sequence with a population of tailed        random primers under hybridization conditions;    -   (b) performing a template-dependent extension reaction that is        primed by a hybridized tailed random primer and that uses the        portion of the target nucleic acid molecule downstream of the        site of hybridization as a template;    -   (c) amplifying a portion of the target nucleic acid molecule and        the tailed random primer sequence with a first tail primer and a        first target-specific primer;    -   (d) amplifying a portion of the amplicon resulting from step (c)        with a second tail primer and a second target-specific primer;    -   (e) sequencing the amplified portion from step (d) using a first        and second sequencing primer;

wherein the population of tailed random primers comprisessingle-stranded oligonucleotide molecules having a 5′ nucleic acidsequence identical to a first sequencing primer; a middle barcodeportion comprising; and a 3′ nucleic acid sequence comprising from about6 to about 12 random nucleotides;

wherein the first target-specific primer comprises a nucleic acidsequence that can specifically anneal to the known target nucleotidesequence of the target nucleic acid at the annealing temperature;

wherein the second target-specific primer comprises a 3′ portioncomprising a nucleic acid sequence that can specifically anneal to aportion of the known target nucleotide sequence comprised by theamplicon resulting from step (c), and a 5′ portion comprising a nucleicacid sequence that is identical to a second sequencing primer and thesecond target-specific primer is nested with respect to the firsttarget-specific primer;

wherein the first tail primer comprises a nucleic acid sequenceidentical to the tailed random primer; and

wherein the second tail primer comprises a nucleic acid sequenceidentical to a portion of the first sequencing primer and is nested withrespect to the first tail primer.

8. The method of paragraph 7, wherein the each tailed random primerfurther comprises a spacer nucleic acid sequence between the 5′ nucleicacid sequence identical to a first sequencing primer and the 3′ nucleicacid sequence comprising about 6 to about 12 random nucleotides.

9. The method of paragraph 7 or 8, wherein the unhybridized primers areremoved from the reaction after an extension step.

10. The method of any of paragraphs 7-9, wherein the second tail primeris nested with respect to the first tail primer by at least 3nucleotides.

11. The method of any of paragraphs 7-10, wherein the firsttarget-specific primer further comprises a 5′ tag sequence portioncomprising a nucleic acid sequence of high GC content which is notsubstantially complementary to or substantially identical to any otherportion of any of the primers.

12. The method of any of paragraphs 7-11, wherein the second tail primeris identical to the full-length first sequencing primer.

13. The method of any of paragraphs 7-12, wherein the portions of thetarget-specific primers that specifically anneal to the known targetwill anneal specifically at a temperature of about 65° C. in a PCRbuffer.

14. The method of any of paragraphs 7-13, wherein the sample comprisesgenomic DNA.

15. The method of any of paragraphs 7-14, wherein the sample comprisesRNA and the method further comprises a first step of subjecting thesample to a reverse transcriptase regimen.

16. The method of any of paragraphs 7-15, wherein the nucleic acidspresent in the sample have not been subjected to shearing or digestion.

17. The method of any of paragraphs 7-16, wherein the sample comprisessingle-stranded gDNA or cDNA. 18. The method of any of paragraphs 7-17,wherein the reverse transcriptase regimen comprises the use of randomhexamers.

19. The method of any of paragraphs 7-18, wherein a gene rearrangementcomprises the known target sequence.

20. The method of paragraph 19, wherein the gene rearrangement ispresent in a nucleic acid selected from the group consisting of: genomicDNA; RNA; and cDNA.

21. The method of any of paragraphs 19-20, wherein the generearrangement comprises an oncogene.

22. The method of paragraph 21, wherein the gene rearrangement comprisesa fusion oncogene.

23. The method of any of paragraphs 7-22, wherein the nucleic acidproduct is sequenced by a next-generation sequencing method.

24. The method of paragraph 23, wherein the next-generation sequencingmethod comprises a method selected from the group consisting of:

Ion Torrent, Illumina, SOLiD, 454; Massively Parallel SignatureSequencing solid-phase, reversible dye-terminator sequencing; and DNAnanoball sequencing.

25. The method of any of paragraphs 7-24, wherein the first and secondsequencing primers are compatible with the selected next-generationsequencing method.

26. The method of any of paragraphs 7-25, wherein the method comprisescontacting the sample, or separate portions of the sample, with aplurality of sets of first and second target-specific primers.

27. The method of any of paragraphs 7-26, wherein the method comprisescontacting a single reaction mixture comprising the sample with aplurality of sets of first and second target-specific primers.

28. The method of any of paragraphs 7-27, wherein the plurality of setsof first and second target-specific primers specifically anneal to knowntarget nucleotide sequences comprised by separate genes.

29. The method of any of paragraphs 7-28, wherein at least two sets offirst and second target-specific primers specifically anneal todifferent portions of a known target nucleotide sequence.

30. The method of any of paragraphs 7-29, wherein at least two sets offirst and second target-specific primers specifically anneal todifferent portions of a single gene comprising a known target nucleotidesequence.

31. The method of any of paragraphs 7-30, wherein at least two sets offirst and second target-specific primers specifically anneal todifferent exons of a gene comprising a known nucleotide target sequence.

32. The method of any of paragraphs 7-31, wherein the plurality of firsttarget-specific primers comprise identical 5′ tag sequence portions.

33. The method of any of paragraphs 7-32, wherein each tailed randomprimer in a population of tailed random primers further comprises anidentical sample barcoding portion.

34. The method of paragraph 33, wherein multiple samples are eachcontacted with a separate population of tailed random primers with asample barcoding portion; wherein each population of tailed randomprimers has a distinct sample barcoding portion; and wherein the samplesare pooled after step (b).

35. The method of any of paragraphs 7-34, wherein each amplificationstep comprises a set of cycles of a PCR amplification regimen from 5cycles to 20 cycles in length.

36. The method of any of paragraphs 7-35, wherein the target-specificprimers and the tail primers are designed such that they willspecifically anneal to their complementary sequences at an annealingtemperature of from about 61 to 72° C.

37. The method of any of paragraphs 7-36, wherein the target-specificprimers and the tail primers are designed such that they willspecifically anneal to their complementary sequences at an annealingtemperature of about 65° C.

38. The method of any of paragraphs 7-37, wherein the target nucleicacid molecule is from a sample, optionally which is a biological sampleobtained from a subject.

39. The method of paragraph 38, wherein the sample is obtained from asubject in need of treatment for a disease associated with a geneticalteration.

40. The method of paragraph 39, wherein the disease is cancer.

41. The method of paragraph 38, wherein the sample comprises apopulation of tumor cells.

42. The method of paragraph 38, wherein the sample is a tumor biopsy.

43. The method paragraph 40, wherein the cancer is lung cancer.

44. The method of any of paragraphs 7-43, wherein a disease-associatedgene comprises the known target sequence.

45. The method of 38, wherein a gene rearrangement product in the samplecomprises the known target sequence.

46. The method of paragraph 45, wherein the gene rearrangement productis an oncogene.

47. A method of preparing nucleic acids for analysis, the methodcomprising:

-   -   (a) contacting a nucleic acid template comprising a first strand        of a target nucleic acid with a complementary target-specific        primer that comprises a target-specific hybridization sequence,        under conditions to promote template-specific hybridization and        extension of the target-specific primer; and    -   (b) contacting a nucleic acid template comprising a second        strand that is complementary to the first strand of the target        nucleic acid with a plurality of different primers that share a        common sequence that is 5′ to different hybridization sequences,        under conditions to promote template-specific hybridization and        extension of at least one of the plurality of different primers,

wherein an extension product is generated to contain both a sequencethat is characteristic of the target-specific primer and a sequence thatis characteristic of the at least one of the plurality of differentprimers.

48. The method of paragraph 47, wherein the target nucleic acid is aribonucleic acid.

49. The method of paragraph 47, wherein the target nucleic acid is adeoxyribonucleic acid.

50. The method of any of paragraphs 47 to 49 wherein steps (a) and (b)are performed sequentially.

51. The method of any of paragraphs 47 to 50, wherein the nucleic acidtemplate in step (a) comprises an extension product resulting from thehybridization and extension of the at least one of the plurality ofdifferent primers in step (b).

52. The method of any one of paragraphs 47 to 50, wherein the nucleicacid template in step (b) comprises an extension product resulting fromthe hybridization and extension of the target-specific primer in step(a).

53. The method of paragraph 48, wherein the target nucleic acid is amessenger RNA encoded from a chromosomal segment that comprises agenetic rearrangement.

54. The method of paragraph 49, wherein the target nucleic acid is achromosomal segment that comprises a portion of a genetic rearrangement.

55. The method of paragraph 8, wherein the genetic rearrangement is aninversion, deletion, or translocation.

56. The method of any one of paragraphs 47 to 55 further comprisingamplifying the extension product.

57. The method of any one of paragraphs 47 to 55 further comprisingcontacting the extension product or amplified extension product with animmobilized oligonucleotide under conditions in which hybridizationoccurs between the extension product and immobilized oligonucleotide.

58. The method of any preceding paragraph wherein the target nucleicacid comprises a target portion having a known sequence and a flankingportion having an unknown sequence.

59. The method of paragraph 58, wherein different hybridizationsequences are complementary to the flanking portion.

60. The method of paragraph 58 or 59, wherein the target-specifichybridization sequence is complementary to the target portion.

61. The method of any of paragraphs 47 to 60, wherein thetarget-specific primer further comprises, 5′ to the target-specifichybridization sequence, at least one of an index sequence, a barcodesequence and an adaptor sequence.

62. The method of any of paragraphs 47 to 60, wherein the commonsequence comprises at least one of an index sequence, barcode sequenceand an adaptor sequence.

63. The method of any of paragraphs 1-62, wherein the adaptor sequenceis a cleavable adaptor sequence for immobilizing oligonucleotides in aflow cell.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

1. A method of preparing nucleic acids for analysis, the methodcomprising:

-   -   (a) contacting a nucleic acid template comprising a first strand        of a target nucleic acid with a complementary target-specific        primer that comprises a target-specific hybridization sequence,        under conditions to promote template-specific hybridization and        extension of the target-specific primer; and    -   (b) contacting a nucleic acid template comprising a second        strand that is complementary to the first strand of the target        nucleic acid with a plurality of different primers that share a        common sequence that is 5′ to different hybridization sequences,        under conditions to promote template-specific hybridization and        extension of at least one of the plurality of different primers,        wherein an extension product is generated to contain both a        sequence that is characteristic of the target-specific primer        and a sequence that is characteristic of the at least one of the        plurality of different primers.

2. The method of paragraph 1, wherein the target nucleic acid is aribonucleic acid.

3. The method of paragraph 1, wherein the target nucleic acid is adeoxyribonucleic acid.

4. The method of any of paragraphs 1 to 3 wherein steps (a) and (b) areperformed sequentially.

5. The method of any of paragraphs 1 to 4, wherein the nucleic acidtemplate in step (a) comprises an extension product resulting from thehybridization and extension of the at least one of the plurality ofdifferent primers in step (b).

6. The method of any one of paragraphs 1 to 4, wherein the nucleic acidtemplate in step (b) comprises an extension product resulting from thehybridization and extension of the target-specific primer in step (a).

7. The method of paragraph 2, wherein the target nucleic acid is amessenger RNA encoded from a chromosomal segment that comprises agenetic rearrangement.

8. The method of paragraph 3, wherein the target nucleic acid is achromosomal segment that comprises a portion of a genetic rearrangement.

9. The method of paragraph 8, wherein the genetic rearrangement is aninversion, deletion, or translocation.

10. The method of any one of paragraphs 1 to 9 further comprisingamplifying the extension product.

11. The method of any one of paragraphs 1 to 9 further comprisingcontacting the extension product or amplified extension product with animmobilized oligonucleotide under conditions in which hybridizationoccurs between the extension product and immobilized oligonucleotide.

12. The method of any preceding paragraph wherein the target nucleicacid comprises a target portion having a known sequence and a flankingportion having an unknown sequence.

13. The method of paragraph 12, wherein different hybridizationsequences are complementary to the flanking portion.

14. The method of paragraph 12 or 13, wherein the target-specifichybridization sequence is complementary to the target portion.

15. The method of any of paragraphs 1 to 14, wherein the target-specificprimer further comprises, 5′ to the target-specific hybridizationsequence, at least one of an index sequence, a barcode sequence and anadaptor sequence.

16. The method of any of paragraphs 1 to 14, wherein the common sequencecomprises at least one of an index sequence, barcode sequence and anadaptor sequence.

17. The method of paragraph 15 or 16, wherein the adaptor sequence is acleavable adaptor sequence for immobilizing oligonucleotides in a flowcell.

18. A method of determining the nucleotide sequence contiguous to aknown target nucleotide sequence, the method comprising;

-   -   (a) contacting a target nucleic acid molecule comprising the        known target nucleotide sequence with an initial target-specific        primer under hybridization conditions;    -   (b) performing a template-dependent extension reaction that is        primed by a hybridized initial target-specific primer and that        uses the target nucleic acid molecule as a template;    -   (c) contacting the product of step (b) with a population of        tailed random primers under hybridization conditions;    -   (d) performing a template-dependent extension reaction that is        primed by a hybridized tailed random primer and that uses the        portion of the target nucleic acid molecule downstream of the        site of hybridization as a template;    -   (e) amplifying a portion of the target nucleic acid molecule and        the tailed random primer sequence with a first tail primer and a        first target-specific primer;    -   (f) amplifying a portion of the amplicon resulting from step (e)        with a second tail primer and a second target-specific primer;    -   (g) sequencing the amplified portion from step (f) using a first        and second sequencing primer;

wherein the population of tailed random primers comprisessingle-stranded oligonucleotide molecules having a 5′ nucleic acidsequence identical to a first sequencing primer and a 3′ nucleic acidsequence comprising from about 6 to about 12 random nucleotides;

wherein the first target-specific primer comprises a nucleic acidsequence that can specifically anneal to the known target nucleotidesequence of the target nucleic acid at the annealing temperature;

wherein the second target-specific primer comprises a 3′ portioncomprising a nucleic acid sequence that can specifically anneal to aportion of the known target nucleotide sequence comprised by theamplicon resulting from step (e), and a 5′ portion comprising a nucleicacid sequence that is identical to a second sequencing primer and thesecond target-specific primer is nested with respect to the firsttarget-specific primer;

wherein the first tail primer comprises a nucleic acid sequenceidentical to the tailed random primer; and

wherein the second tail primer comprises a nucleic acid sequenceidentical to a portion of the first sequencing primer and is nested withrespect to the first tail primer.

19. A method of determining the nucleotide sequence contiguous to aknown target nucleotide sequence, the method comprising;

-   -   (a) contacting a target nucleic acid molecule comprising the        known target nucleotide sequence with a population of tailed        random primers under hybridization conditions;    -   (b) performing a template-dependent extension reaction that is        primed by a hybridized tailed random primer and that uses the        portion of the target nucleic acid molecule downstream of the        site of hybridization as a template;    -   (c) contacting the product of step (b) with an initial        target-specific primer under hybridization conditions;    -   (d) performing a template-dependent extension reaction that is        primed by a hybridized initial target-specific primer and that        uses the target nucleic acid molecule as a template;    -   (e) amplifying a portion of the target nucleic acid molecule and        the tailed random primer sequence with a first tail primer and a        first target-specific primer;    -   (f) amplifying a portion of the amplicon resulting from step (e)        with a second tail primer and a second target-specific primer;    -   (g) sequencing the amplified portion from step (f) using a first        and second sequencing primer;

wherein the population of tailed random primers comprisessingle-stranded oligonucleotide molecules having a 5′ nucleic acidsequence identical to a first sequencing primer and a 3′ nucleic acidsequence comprising from about 6 to about 12 random nucleotides;

wherein the first target-specific primer comprises a nucleic acidsequence that can specifically anneal to the known target nucleotidesequence of the target nucleic acid at the annealing temperature;

wherein the second target-specific primer comprises a 3′ portioncomprising a nucleic acid sequence that can specifically anneal to aportion of the known target nucleotide sequence comprised by theamplicon resulting from step (c), and a 5′ portion comprising a nucleicacid sequence that is identical to a second sequencing primer and thesecond target-specific primer is nested with respect to the firsttarget-specific primer;

wherein the first tail primer comprises a nucleic acid sequenceidentical to the tailed random primer; and

wherein the second tail primer comprises a nucleic acid sequenceidentical to a portion of the first sequencing primer and is nested withrespect to the first tail primer.

20. The method of any of paragraphs 18-19, further comprising a step ofcontacting the sample and products with RNase after extension of theinitial target-specific primer.

21. The method of any of paragraphs 1-3, wherein the tailed randomprimer can form a hair-pin loop structure.

22. The method of any of paragraphs 1-4, wherein the initialtarget-specific primer and the first target-specific primer areidentical.

23. The method of any of paragraphs 1-5, wherein the tailed randomprimer further comprises a barcode portion comprising 6-12 randomnucleotides between the 5′ nucleic acid sequence identical to a firstsequencing primer and the 3′ nucleic acid sequence comprising 6-12random nucleotides. 7. A method of determining the nucleotide sequencecontiguous to a known target nucleotide sequence, the method comprising;

-   -   (a) contacting a target nucleic acid molecule comprising the        known target nucleotide sequence with a population of tailed        random primers under hybridization conditions;    -   (b) performing a template-dependent extension reaction that is        primed by a hybridized tailed random primer and that uses the        portion of the target nucleic acid molecule downstream of the        site of hybridization as a template;    -   (c) amplifying a portion of the target nucleic acid molecule and        the tailed random primer sequence with a first tail primer and a        first target-specific primer;    -   (d) amplifying a portion of the amplicon resulting from step (c)        with a second tail primer and a second target-specific primer;    -   (e) sequencing the amplified portion from step (d) using a first        and second sequencing primer;

wherein the population of tailed random primers comprisessingle-stranded oligonucleotide molecules having a 5′ nucleic acidsequence identical to a first sequencing primer; a middle barcodeportion comprising; and a 3′ nucleic acid sequence comprising from about6 to about 12 random nucleotides;

wherein the first target-specific primer comprises a nucleic acidsequence that can specifically anneal to the known target nucleotidesequence of the target nucleic acid at the annealing temperature;

wherein the second target-specific primer comprises a 3′ portioncomprising a nucleic acid sequence that can specifically anneal to aportion of the known target nucleotide sequence comprised by theamplicon resulting from step (c), and a 5′ portion comprising a nucleicacid sequence that is identical to a second sequencing primer and thesecond target-specific primer is nested with respect to the firsttarget-specific primer;

wherein the first tail primer comprises a nucleic acid sequenceidentical to the tailed random primer; and

wherein the second tail primer comprises a nucleic acid sequenceidentical to a portion of the first sequencing primer and is nested withrespect to the first tail primer.

24. The method of paragraph 23, wherein the each tailed random primerfurther comprises a spacer nucleic acid sequence between the 5′ nucleicacid sequence identical to a first sequencing primer and the 3′ nucleicacid sequence comprising about 6 to about 12 random nucleotides.

25. The method of paragraph 23 or 24, wherein the unhybridized primersare removed from the reaction after an extension step.

26. The method of any of paragraphs 23-25, wherein the second tailprimer is nested with respect to the first tail primer by at least 3nucleotides.

27. The method of any of paragraphs 23-26, wherein the firsttarget-specific primer further comprises a 5′ tag sequence portioncomprising a nucleic acid sequence of high GC content which is notsubstantially complementary to or substantially identical to any otherportion of any of the primers.

28. The method of any of paragraphs 23-27, wherein the second tailprimer is identical to the full-length first sequencing primer.

29. The method of any of paragraphs 23-28, wherein the portions of thetarget-specific primers that specifically anneal to the known targetwill anneal specifically at a temperature of about 65° C. in a PCRbuffer.

30. The method of any of paragraphs 23-29, wherein the sample comprisesgenomic DNA.

31. The method of any of paragraphs 23-30, wherein the sample comprisesRNA and the method further comprises a first step of subjecting thesample to a reverse transcriptase regimen.

32. The method of any of paragraphs 23-31, wherein the nucleic acidspresent in the sample have not been subjected to shearing or digestionor wherein the sample comprises single-stranded gDNA or cDNA.

33. The method of any of paragraphs 23-32, wherein the reversetranscriptase regimen comprises the use of random hexamers.

34. The method of any of paragraphs 23-33, wherein a gene rearrangementcomprises the known target sequence.

35. The method of paragraph 34, wherein the gene rearrangement ispresent in a nucleic acid selected from the group consisting of: genomicDNA; RNA; and cDNA.

36. The method of any of paragraphs 34-35, wherein the generearrangement comprises an oncogene.

37. The method of paragraph 36, wherein the gene rearrangement comprisesa fusion oncogene.

38. The method of any of paragraphs 23-37, wherein the nucleic acidproduct is sequenced by a next-generation sequencing method.

39. The method of paragraph 38, wherein the next-generation sequencingmethod comprises a method selected from the group consisting of:

Ion Torrent, Illumina, SOLiD, 454; Massively Parallel SignatureSequencing solid-phase, reversible dye-terminator sequencing; and DNAnanoball sequencing.

40. The method of any of paragraphs 23-39, wherein the first and secondsequencing primers are compatible with the selected next-generationsequencing method.

41. The method of any of paragraphs 23-40, wherein the method comprisescontacting the sample, or separate portions of the sample, with aplurality of sets of first and second target-specific primers.

42. The method of any of paragraphs 23-41, wherein the method comprisescontacting a single reaction mixture comprising the sample with aplurality of sets of first and second target-specific primers.

43. The method of any of paragraphs 23-42, wherein the plurality of setsof first and second target-specific primers specifically anneal to knowntarget nucleotide sequences comprised by separate genes.

44. The method of any of paragraphs 23-43, wherein at least two sets offirst and second target-specific primers specifically anneal todifferent portions of a known target nucleotide sequence.

45. The method of any of paragraphs 23-44, wherein at least two sets offirst and second target-specific primers specifically anneal todifferent portions of a single gene comprising a known target nucleotidesequence.

46. The method of any of paragraphs 23-45, wherein at least two sets offirst and second target-specific primers specifically anneal todifferent exons of a gene comprising a known nucleotide target sequence.

47. The method of any of paragraphs 23-46, wherein the plurality offirst target-specific primers comprise identical 5′ tag sequenceportions.

48. The method of any of paragraphs 23-47, wherein each tailed randomprimer in a population of tailed random primers further comprises anidentical sample barcoding portion.

49. The method of paragraph 48, wherein multiple samples are eachcontacted with a separate population of tailed random primers with asample barcoding portion; wherein each population of tailed randomprimers has a distinct sample barcoding portion; and wherein the samplesare pooled after step (b).

50. The method of any of paragraphs 23-49, wherein each amplificationstep comprises a set of cycles of a PCR amplification regimen from 5cycles to 20 cycles in length.

51. The method of any of paragraphs 23-50, wherein the target-specificprimers and the tail primers are designed such that they willspecifically anneal to their complementary sequences at an annealingtemperature of from about 61 to 72° C.

52. The method of any of paragraphs 23-51, wherein the target-specificprimers and the tail primers are designed such that they willspecifically anneal to their complementary sequences at an annealingtemperature of about 65° C.

53. The method of any of paragraphs 23-52, wherein the target nucleicacid molecule is from a sample, optionally which is a biological sampleobtained from a subject.

54. The method of paragraph 53, wherein the sample is obtained from asubject in need of treatment for a disease associated with a geneticalteration.

55. The method of paragraph 54, wherein the disease is cancer.

56. The method of paragraph 53, wherein the sample comprises apopulation of tumor cells.

57. The method of paragraph 53, wherein the sample is a tumor biopsy.

58. The method paragraph 55, wherein the cancer is lung cancer.

59. The method of any of paragraphs 23-58, wherein a disease-associatedgene comprises the known target sequence.

60. The method of 53, wherein a gene rearrangement product in the samplecomprises the known target sequence.

61. The method of paragraph 60, wherein the gene rearrangement productis an oncogene.

EXAMPLES Example 1: A Method Using Reverse Transcriptase with TailedRandom Oligonucleotides and Gene-Specific Oligonucleotides to Amplify 3′Fusion Events

First Strand Synthesis

As a first step towards amplifying 3′ fusion events for sequenceanalysis, RNA was obtained from a sample isolated from a subject. Thefollowing reaction was assembled on ice to synthesize the first cDNAstrand:

-   -   12 μL purified RNA and H2O    -   2 μL 1.2 μg/μL random primer #1 (9 mer)    -   2 μL dNTP        The reaction was transferred to a thermocycler and incubated at        65° C. for 5 minutes. Then, the reaction was centrifuged and        incubated on ice for at least one minute.

To the reaction above, the following was prepared on ice:

-   -   2 μL 10× M-MuLV reverse transcriptase buffer    -   1 μL 40 U/μL RNase inhibitor    -   1 μL 200 U/μL M-MuLV enzyme        The reaction was mixed and centrifuged briefly to collect the        reaction contents at the bottom of the tube, then placed back on        ice. The reaction was then incubated at 42° C. for 60 minutes,        followed by 4° C.

ExoI Treatment

The following was added to the reaction above,

-   -   1 μL 20 U/μL Exonuclease I

The reaction was mixed and centrifuged briefly to collect the contentsat the bottom of the tube, then incubated at 37° C. for 10 minutes.Next, 1.28 μL of 1N NaOH was added and mixed by pipetting up and downthen centrifuged to collect the contents. The reaction was incubated at80° C. for 10 minutes, then 4 μL 10 mM Tris pH 8.3 was added ad mixed bypipetting up and down. 20 μL of the Exonuclease-treated DNA solution wastransferred to a fresh 200 μL PCR tube on ice.

Second Strand cDNA Synthesis

The following reaction was prepared:

-   -   20 μL DNA solution, from above    -   11 μL nuclease-free H₂O    -   4 μL 10×PCR Buffer II    -   4 μL 3 μM gene-specific primer #1    -   1 μL 0.5 mM dNTP

The reaction was mixed by pipetting up and down then centrifuged brieflyto collect the contents and placed on ice. The reaction was thenincubated at 95° C. for 3 minutes, then 22° C. for 10 seconds followedby 4° C. until proceeding to the next step. The reaction was incubatedon ice for at least one minutes.

The following was added to the reaction:

-   -   1 μL 400 U/μL Manta 1.0 DNA Polymerase (high concentration)

The reaction was incubated at 25° C. for 10 seconds, then 70° C. for 10minutes and maintained at 4° C. until proceeding to the next step.

DNA Purification with AMPure Beads #1

The following was added to the reaction above,

-   -   88.4 μL AMPure beads

The suspension was mixed well and incubated for 5 minutes at roomtemperature. A magnet was used 2-4 minutes to collect the beads and thesolution appeared clear. The supernatant was discarded and the beadswere washed twice times with 200 μL 70% ethanol on the magnet. After thesecond wash, the beads were dried at room temperature for 5 minutes.Finally, the DNA was eluted by removing the tubes from the magnet andresuspending the beads in 12 μL 10 mM Tris-HCl pH 8.3 elution bufferincluded in the AMPure kit. The RNA-bead solution was placed on themagnet for 2 minutes. Then, the DNA solution was transferred to a freshPCR tube, being sure to avoid transferring beads to the fresh tube.

It should be appreciated that in some embodiments the ratio of beads toreaction mix can effect the size of fragments returned. In someembodiments, for fusion detection, all or substantially all fragmentsare longer than 60 nt (e.g., 30 nt on either side of a fusion breakpoint or junction) so each gene can be easily identified.

Amplification #1

The following reaction was prepared:

-   -   10 μL purified DNA, from Purification #1 above    -   4 μL 5× Phoenix Hot Start Buffer    -   2 μL 2 mM dNTP    -   2 μL 10 μM P5_barcode primer    -   2 μL 3 μM gene-specific primer #1    -   0.5 to 2 Units polymerase (e.g., Pheonix Hot Start Taq, VeraSeq)

The reaction was incubated as follows:

-   -   Step 1: 95° C. for 3 minutes    -   Step 2: 95° C. for 30 seconds    -   Step 3: 65° C. for 5 minutes, return to step 2 for 14 cycles    -   Step 4: 72° C. for 2 minutes    -   Step 5: 4° C., until proceeding with the protocol

DNA Purification with AMPure Beads #2

The following was added to the reaction above,

-   -   36.4 μL AMPure beads

The suspension was mixed well and incubated for 5 minutes at roomtemperature. A magnet was used 2-4 minutes to collect the beads and thesolution appeared clear. The supernatant was discarded and the beadswere washed twice times with 200 μL 70% ethanol on the magnet. After thesecond wash, the beads were dried at room temperature for 5 minutes.Finally, the DNA was eluted by removing the tubes from the magnet andresuspending the beads in 9 μL 10 mM Tris-HCl pH 8.3 elution bufferincluded in the AMPure kit. The RNA-bead solution was placed on themagnet for 2 minutes. Then, the DNA solution was transferred to a freshPCR tube, being sure to avoid transferring beads to the fresh tube.

Amplification #2

The following reaction was prepared:

-   -   8.5 μL purified DNA, from Purification #2 above    -   4 μL 5× Phoenix Hot Start Buffer    -   2 μL 2 mM dNTP    -   2 μL 10 μM P5_29 bp primer    -   2 μL 10 μM P7 barcode primer    -   2 μL 3 μM gene-specific primer #2    -   0.2 μL 5 U/μL Phoenix Hot Start Taq polymerase    -   2 μL 10 μM P7 barcode primer

The reaction was incubated as follows:

-   -   Step 1: 95° C. for 3 minutes    -   Step 2: 95° C. for 30 seconds    -   Step 3: 65° C. for 5 minutes, return to step 2 for 14 cycles    -   Step 4: 72° C. for 2 minutes    -   Step 5: 4° C., until proceeding with the protocol

DNA Purification with AMPure Beads #3

The following was added to the reaction above,

-   -   37.3 μL AMPure beads

The suspension was mixed well and incubated for 5 minutes at roomtemperature. A magnet was used 2-4 minutes to collect the beads and thesolution appeared clear. The supernatant was discarded and the beadswere washed twice times with 200 μL 70% ethanol on the magnet. After thesecond wash, the beads were dried at room temperature for 5 minutes.Finally, the DNA was eluted by removing the tubes from the magnet andresuspending the beads in 20 μL 10 mM Tris-HCl pH 8.3 elution bufferincluded in the AMPure kit. The RNA-bead solution was placed on themagnet for 2 minutes. Then, the DNA solution was transferred to a freshPCR tube, being sure to avoid transferring beads to the fresh tube.

Quantification of Library Concentration

The Kapa Biosystems qPCR kit for Illumina was used to quantitate theconcentration of each library prepared using the protocol above. Thebarcoded libraries were pooled at equimolar concentrations. Then thelibrary was loaded on an Illumina MiSeq at XpM using the MiSeq v2 300cycle reagent kit following the manufacturer's instruction. The sampleswere sequenced using 2×150 bp reads with 7 base encoded index reads.

Example 2: A Method Using Reverse Transcriptase with Gene-SpecificOligonucleotides and Tailed Random Oligonucleotides to Amplify 5′ FusionEvents

First Strand Synthesis

As a first step towards amplifying 5′ fusion events for sequenceanalysis, RNA was obtained from a sample isolated from a subject. Thefollowing reaction was assembled on ice to synthesize the first cDNAstrand:

-   -   12 μL purified RNA and H2O    -   2 μL gene-specific primer #1    -   2 μL dNTP

The reaction was transferred to a thermocycler and incubated at 65° C.for 5 minutes. Then, the reaction was centrifuged and incubated on icefor at least one minute.

To the reaction above, the following was prepared on ice:

-   -   2 μL 10× M-MuLV reverse transcriptase buffer    -   1 μL 40 U/μL RNase inhibitor    -   1 μL 200 U/μL M-MuLV enzyme

The reaction was mixed and centrifuged briefly to collect the reactioncontents at the bottom of the tube, then placed back on ice. Thereaction was then incubated at 42° C. for 60 minutes, followed by 4° C.

ExoI Treatment

The following was added to the reaction above,

-   -   1 μL 20 U/μL Exonuclease I

The reaction was mixed and centrifuged briefly to collect the contentsat the bottom of the tube, then incubated at 37° C. for 10 minutes.Next, 1.28 μL of 1N NaOH was added and mixed by pipetting up and downthen centrifuged to collect the contents. The reaction was incubated at80° C. for 10 minutes, then 4 μL 10 mM Tris pH 8.3 was added ad mixed bypipetting up and down. 20 μL of the Exonuclease-treated DNA solution wastransferred to a fresh 200 μL PCR tube on ice.

Second Strand cDNA Synthesis

The following reaction was prepared:

-   -   20 μL DNA solution, from above    -   11 μL nuclease-free H₂O    -   4 μL 10×PCR Buffer II    -   4 μL 1.2 μg/μL random primer (9 mer)    -   1 μL 0.5 mM dNTP

The reaction was mixed by pipetting up and down then centrifuged brieflyto collect the contents and placed on ice. The reaction was thenincubated at 95° C. for 3 minutes, then 22° C. for 10 seconds followedby 4° C. until proceeding to the next step. The reaction was incubatedon ice for at least one minutes.

The following was added to the reaction:

-   -   1 μL 400 U/μL Manta 1.0 DNA Polymerase (high concentration)

The reaction was incubated at 25° C. for 10 seconds, then 70° C. for 10minutes and maintained at 4° C. until proceeding to the next step.

DNA Purification with AMPure Beads #1

The following was added to the reaction above,

-   -   88.4 μL AMPure beads

The suspension was mixed well and incubated for 5 minutes at roomtemperature. A magnet was used 2-4 minutes to collect the beads and thesolution appeared clear. The supernatant was discarded and the beadswere washed twice times with 200 μL 70% ethanol on the magnet. After thesecond wash, the beads were dried at room temperature for 5 minutes.Finally, the DNA was eluted by removing the tubes from the magnet andresuspending the beads in 12 μL 10 mM Tris-HCl pH 8.3 elution bufferincluded in the AMPure kit. The RNA-bead solution was placed on themagnet for 2 minutes. Then, the DNA solution was transferred to a freshPCR tube, being sure to avoid transferring beads to the fresh tube.

Amplification #1

The following reaction was prepared:

-   -   10 μL purified DNA, from Purification #1 above    -   4 μL 5× Phoenix Hot Start Buffer    -   2 μL 2 mM dNTP    -   2 μL 10 μM P5_barcode primer    -   2 μL 3 μM gene-specific primer #1    -   0.5 to 2 Units polymerase (e.g., Pheonix Hot Start Taq, VeraSeq)

The reaction was incubated as follows:

-   -   Step 1: 95° C. for 3 minutes    -   Step 2: 95° C. for 30 seconds    -   Step 3: 65° C. for 5 minutes, return to step 2 for 14 cycles    -   Step 4: 72° C. for 2 minutes    -   Step 5: 4° C., until proceeding with the protocol

DNA Purification with AMPure Beads #2

The following was added to the reaction above,

-   -   36.4 μL AMPure beads

The suspension was mixed well and incubated for 5 minutes at roomtemperature. A magnet was used 2-4 minutes to collect the beads and thesolution appeared clear. The supernatant was discarded and the beadswere washed twice times with 200 μL 70% ethanol on the magnet. After thesecond wash, the beads were dried at room temperature for 5 minutes.Finally, the DNA was eluted by removing the tubes from the magnet andresuspending the beads in 9 μL 10 mM Tris-HCl pH 8.3 elution bufferincluded in the AMPure kit. The RNA-bead solution was placed on themagnet for 2 minutes. Then, the DNA solution was transferred to a freshPCR tube, being sure to avoid transferring beads to the fresh tube.

Amplification #2

The following reaction was prepared:

-   -   8.5 μL purified DNA, from Purification #2 above    -   4 μL 5× Phoenix Hot Start Buffer    -   2 μL 2 mM dNTP    -   2 μL 10 μM P5_29 bp primer    -   2 μL 10 μM P7 barcode primer    -   2 μL 3 μM gene-specific primer #2    -   0.2 μL 5 U/μL Phoenix Hot Start Taq polymerase    -   2 μL 10 μM P7 barcode primer

The reaction was incubated as follows:

-   -   Step 1: 95° C. for 3 minutes    -   Step 2: 95° C. for 30 seconds    -   Step 3: 65° C. for 5 minutes, return to step 2 for 14 cycles    -   Step 4: 72° C. for 2 minutes    -   Step 5: 4° C., until proceeding with the protocol

DNA Purification with AMPure Beads #3

The following was added to the reaction above,

-   -   37.3 μL AMPure beads

The suspension was mixed well and incubated for 5 minutes at roomtemperature. A magnet was used 2-4 minutes to collect the beads and thesolution appeared clear. The supernatant was discarded and the beadswere washed twice times with 200 μL 70% ethanol on the magnet. After thesecond wash, the beads were dried at room temperature for 5 minutes.Finally, the DNA was eluted by removing the tubes from the magnet andresuspending the beads in 20 μL 10 mM Tris-HCl pH 8.3 elution bufferincluded in the AMPure kit. The RNA-bead solution was placed on themagnet for 2 minutes. Then, the DNA solution was transferred to a freshPCR tube, being sure to avoid transferring beads to the fresh tube.

Quantification of Library Concentration

The Kapa Biosystems qPCR kit for Illumina was used to quantitate theconcentration of each library prepared using the protocol above. Thebarcoded libraries were pooled at equimolar concentrations. Then thelibrary was loaded on an Illumina MiSeq at XpM using the MiSeq v2 300cycle reagent kit following the manufacturer's instruction. The sampleswere sequenced using 2×150 bp reads with 7 base encoded index reads.

What is claimed herein is:
 1. A method of preparing nucleic acids foranalysis, the method comprising: (a) contacting a nucleic acid templatecomprising a first strand of a target nucleic acid with a complementarytarget-specific primer that comprises a target-specific hybridizationsequence, under conditions to promote template-specific hybridizationand extension of the target-specific primer; and (b) contacting anucleic acid template comprising a second strand that is complementaryto the first strand of the target nucleic acid with a plurality ofdifferent primers that share a common sequence that is 5′ to differenthybridization sequences, under conditions to promote template-specifichybridization and extension of at least one of the plurality ofdifferent primers, wherein an extension product is generated to containboth a sequence that is characteristic of the target-specific primer anda sequence that is characteristic of the at least one of the pluralityof different primers.
 2. The method of claim 1, wherein the targetnucleic acid is a ribonucleic acid.
 3. The method of claim 1, whereinthe target nucleic acid is a deoxyribonucleic acid.
 4. The method ofclaim 1, wherein steps (a) and (b) are performed sequentially.
 5. Themethod of claim 1, wherein the nucleic acid template in step (a)comprises an extension product resulting from the hybridization andextension of the at least one of the plurality of different primers instep (b).
 6. The method of claim 1, wherein the nucleic acid template instep (b) comprises an extension product resulting from the hybridizationand extension of the target-specific primer in step (a).
 7. The methodof claim 2, wherein the target nucleic acid is a messenger RNA encodedfrom a chromosomal segment that comprises a genetic rearrangement. 8.The method of claim 3, wherein the target nucleic acid is a chromosomalsegment that comprises a portion of a genetic rearrangement.
 9. Themethod of claim 8, wherein the genetic rearrangement is an inversion,deletion, or translocation.
 10. The method of claim 1, furthercomprising amplifying the extension product.
 11. The method of claim 1,further comprising contacting the extension product or amplifiedextension product with an immobilized oligonucleotide under conditionsin which hybridization occurs between the extension product andimmobilized oligonucleotide.
 12. The method of claim 1 wherein thetarget nucleic acid comprises a target portion having a known sequenceand a flanking portion having an unknown sequence.
 13. The method ofclaim 12, wherein different hybridization sequences are complementary tothe flanking portion.
 14. The method of claim 12, wherein thetarget-specific hybridization sequence is complementary to the targetportion.
 15. The method of claim 1, wherein the target-specific primerfurther comprises, 5′ to the target-specific hybridization sequence, atleast one of an index sequence, a barcode sequence and an adaptorsequence.
 16. The method of claim 1, wherein the common sequencecomprises at least one of an index sequence, barcode sequence and anadaptor sequence.
 17. The method of claim 15, wherein the adaptorsequence is a cleavable adaptor sequence for immobilizingoligonucleotides in a flow cell.
 18. A method of determining thenucleotide sequence contiguous to a known target nucleotide sequence,the method comprising; (a) contacting a target nucleic acid moleculecomprising the known target nucleotide sequence with an initialtarget-specific primer under hybridization conditions; (b) performing atemplate-dependent extension reaction that is primed by a hybridizedinitial target-specific primer and that uses the target nucleic acidmolecule as a template; (c) contacting the product of step (b) with apopulation of tailed random primers under hybridization conditions; (d)performing a template-dependent extension reaction that is primed by ahybridized tailed random primer and that uses the portion of the targetnucleic acid molecule downstream of the site of hybridization as atemplate; (e) amplifying a portion of the target nucleic acid moleculeand the tailed random primer sequence with a first tail primer and afirst target-specific primer; (f) amplifying a portion of the ampliconresulting from step (e) with a second tail primer and a secondtarget-specific primer; (g) sequencing the amplified portion from step(f) using a first and second sequencing primer; wherein the populationof tailed random primers comprises single-stranded oligonucleotidemolecules having a 5′ nucleic acid sequence identical to a firstsequencing primer and a 3′ nucleic acid sequence comprising from about 6to about 12 random nucleotides; wherein the first target-specific primercomprises a nucleic acid sequence that can specifically anneal to theknown target nucleotide sequence of the target nucleic acid at theannealing temperature; wherein the second target-specific primercomprises a 3′ portion comprising a nucleic acid sequence that canspecifically anneal to a portion of the known target nucleotide sequencecomprised by the amplicon resulting from step (e), and a 5′ portioncomprising a nucleic acid sequence that is identical to a secondsequencing primer and the second target-specific primer is nested withrespect to the first target-specific primer; wherein the first tailprimer comprises a nucleic acid sequence identical to the tailed randomprimer; and wherein the second tail primer comprises a nucleic acidsequence identical to a portion of the first sequencing primer and isnested with respect to the first tail primer.
 19. A method ofdetermining the nucleotide sequence contiguous to a known targetnucleotide sequence, the method comprising; (a) contacting a targetnucleic acid molecule comprising the known target nucleotide sequencewith a population of tailed random primers under hybridizationconditions; (b) performing a template-dependent extension reaction thatis primed by a hybridized tailed random primer and that uses the portionof the target nucleic acid molecule downstream of the site ofhybridization as a template; (c) contacting the product of step (b) withan initial target-specific primer under hybridization conditions; (d)performing a template-dependent extension reaction that is primed by ahybridized initial target-specific primer and that uses the targetnucleic acid molecule as a template; (e) amplifying a portion of thetarget nucleic acid molecule and the tailed random primer sequence witha first tail primer and a first target-specific primer; (f) amplifying aportion of the amplicon resulting from step (e) with a second tailprimer and a second target-specific primer; (g) sequencing the amplifiedportion from step (f) using a first and second sequencing primer;wherein the population of tailed random primers comprisessingle-stranded oligonucleotide molecules having a 5′ nucleic acidsequence identical to a first sequencing primer and a 3′ nucleic acidsequence comprising from about 6 to about 12 random nucleotides; whereinthe first target-specific primer comprises a nucleic acid sequence thatcan specifically anneal to the known target nucleotide sequence of thetarget nucleic acid at the annealing temperature; wherein the secondtarget-specific primer comprises a 3′ portion comprising a nucleic acidsequence that can specifically anneal to a portion of the known targetnucleotide sequence comprised by the amplicon resulting from step (c),and a 5′ portion comprising a nucleic acid sequence that is identical toa second sequencing primer and the second target-specific primer isnested with respect to the first target-specific primer; wherein thefirst tail primer comprises a nucleic acid sequence identical to thetailed random primer; and wherein the second tail primer comprises anucleic acid sequence identical to a portion of the first sequencingprimer and is nested with respect to the first tail primer.
 20. Themethod of claim 18, further comprising a step of contacting the sampleand products with RNase after extension of the initial target-specificprimer.